Methods for obtaining plant varieties

ABSTRACT

The present invention relates to an isolated and purified DNA comprising a nucleotide sequence that encodes a polypeptide functionally involved in the DNA mismatch repair system of a plant.

TECHNICAL FIELD

[0001] The present invention relates to nucleotide sequences whichencode polypeptides involved in the DNA mismatch repair systems ofplants, and to the polypeptides encoded by those nucleotide sequences.The invention also relates to nucleotide sequences and polypeptidesequences for use in altering the DNA mismatch repair system in plants.The invention also relates to a process for altering the DNA mismatchrepair system of a plant cell, to a process for increasing geneticvariations in plants and to processes for obtaining plants having adesired characteristic.

BACKGROUND OF THE INVENTION

[0002] Plant breeding essentially relies on and makes use of geneticvariation which occurs naturally within and between members of a family,a genus, a species or a subspecies. Another source of genetic variationis the introduction of genes from other organisms which may or may notbe related to the host plant.

[0003] Allelic loci or non-allelic genes which constitute or contributeto desired quantitative (e.g. growth performance, yield. etc.) orqualitative (e.g. deposition, content and composition of seed storageproducts; pathogen resistance genes: etc.) traits that are absent,incomplete or inefficient in a species or subspecies of interest aretypically introduced by the plant breeder from other species orsubspecies. or de novo. This introduction is often done by crossing,provided that the species to be crossed are sexually compatible. Othermeans of introducing genomes, individual chromosomes or genes into plantcells or plants are well known in the art. They include cell fusion,chemically aided transfection (Schocher et al., 1986, Biotechnology 4:1093) and ballistic (McCabe et al., 1988, Biotechnology 6: 923).microinjection (Neuhaus et al., 1987, TAG 75: 30), electroporation ofprotoplasts (Chupeau et al., 1989, Biotechnology 7: 53) or microbialtransformation methods such as Agrobacterium mediated transformation(Horsch et al., 1985, Science 227: 1229; Hiei et al., 1996,Biotechnology 14: 745).

[0004] However, when a foreign genome, chromosome or gene is introducedinto a plant, it will often segregate in subsequent generations from thegenome of the recipient plant or plant cell during mitotic and meioticcell divisions and, in consequence, become lost from the host plant orplant cell into which it had been introduced. Occasionally, however, theintroduced genome, chromosome or gene physically combines entirely or inpart with the genome, chromosome or gene of the host plant or plant cellin a process which is called recombination.

[0005] Recombination involves the exchange of covalent linkages betweenDNA molecules in regions of identical or similar sequence. It isreferred to here as homologous recombination if donor and recipient DNAare identical or nearly identical (at least 99% base sequence identity),and as homeologous recombination if donor and recipient DNA are notidentical but are similar (less than 99% base sequence identity).

[0006] The ability of two genomes, chromosomes or genes to recombine isknown to depend largely on the evolutionary relation between them andthus on the degree of sequence similarity between the two DNA molecules.Whereas homologous recombination is frequently observed during mitosisand meiosis, homeologous recombination is rarely or never seen.

[0007] From a breeder's perspective, the limits within which homologousrecombination occurs, therefore, define a genetic barrier betweenspecies, varieties or lines, in contrast homeologous recombination whichcan break this barrier. Homeologous recombination is thus of greatimportance for plant breeding. Accordingly there is a need for a processfor enhancing the frequency of homeologous recombination in plants. Inparticular, there is a need for a process of increasing homeologousrecombination to significantly shorten the length of breeding programsby reducing the number of crosses required to obtain an otherwise rarerecombination event.

[0008] At least in Escherichia coli, homologous and homeologousrecombination are known to share a common pathway that requires amongothers the proteins RecA, RecB, RecC. RecD and makes use of the SOSinduced RuvA and RuvB, respectively. It has been suggested that matinginduced recombination follows the Double-Strand Break Repair model(Szostak et al., 1983, Cell 33, 25-35), which is widely used to describegenetic recombination in eukaryotes. Following the alignment ofhomologous or homeologous DNA double helices the RecA protein mediatesan exchange of a single DNA strand from the donor helix to the alignedrecipient DNA helix. The incoming strand screens the recipient helix forsequence complementarity, seeking to form a heteroduplex by hydrogenbonding the complementary strand. The displaced homologous orhomeologous strand of the recipient helix is guided into the donor helixwhere it base pairs with its counterpart strand to form a secondheteroduplex. The resulting branch point then migrates along the alignedchromosomes thereby elongating and thus stabilising the initialheteroduplexes. Single stranded gaps (if present) are closed by DNAsynthesis. The strand cross overs (Holliday junction) are eventuallyresolved enzymatically to yield the recombination products.

[0009] Although in wild type E. coli homologous and homeologousrecombination are thus mechanistically similar if not identical,homologous recombination in conjugational crosses E. coli×E. coli occursfive orders of magnitude more frequently than homeologous recombinationin conjugational crosses E. coli×S. typhimurium (Matic et al. 1995; Cell80, 507-515). The imbalance in favour of homologous recombination wasshown to be caused largely by the bacterial MisMatch Repair (MMR) systemsince its inactivation increased the frequency of homeologousrecombination in E. coli up to 1000 fold (Rayssiguier et al. 1989.Nature 342. 396-401).

[0010] In E. Coli, the MMR system (reviewed by Modrich 1991, Annual RevGenetics 25, 229-253) is composed of only three proteins known as MutS,MutL and MutH. MutS recognizes and binds to base pair mismatches. MutLthen forms a stable complex with mismatch bound MutS. This proteincomplex now activates the MutH intrinsic single stranded endonucleasewhich nicks the strand containing the misplaced base and therebyprepares the template for DNA repair enzymes.

[0011] During recombination, MMR components inhibit homeologousrecombination. In vitro experiments demonstrated that MutS in complexwith MutL binds to mismatches at the recombination branch point andphysically blocks RecA mediated strand exchange and heteroduplexformation (Worth et al., 1994; PNAS 91, 3238-3241). Interestingly, theSOS dependent RuvAB mediated branch migration is insensitive toMutS/MutL, explaining the observed slight increase in SOS dependenthomeologous recombination. Homeologous mating even induces the SOSresponse, thereby taking advantage of RuvAB induction (Matic et al.1995. Cell 80. 507-515).

[0012] The MMR system thus appears to be a genetic guardian over genomestability in E. coli. In this role it essentially determines the extentto which genetic isolation, that is, speciation occurs. The diminishedsensitivity of the SOS system to MMR, however, allows (within limits)for rapid genomic changes at times of stress, providing the means forfast adaptation to altered environmental conditions and thuscontributing to intraspecies genetic variation and species evolution.

[0013] The important role of MMR in preserving genomic integrity hasbeen established also in certain eukaryotes. In its efficiency, thehuman MMR, for example, may even counteract potential gene therapy toolssuch as triple-helix forming oligonucleotides including RNA-DNA hybridmolecules (Havre et al., 1993, J. Virology 67: 7234-7331; Wang et al.,1995, Mol. Cell. Biol. 15: 1759-1768; Kotani et al., 1996, Mol. Gen.Genetics 250: 626-634; Cole-Strauss et al., 1996, Science 273:1387-1389). Such oligonucleotides are designed to introduce single basechanges into selected DNA target sequences in order to inactivate forexample cancer genes or to restore their normal function. The resultingbase mismatches however are recognised by the mismatch repair systemwhich then directs removal of the mismatched base, thereby reducing theefficiency of oligonucleotide induced site-specific mutagenesis.

[0014] To date, two families of related genes, homologous to thebacterial MutS and MutL genes have been identified or isolated in yeastand mammals (recent reviews by Arnheim and Shibata, 1997, Curr. OpinionGenet. Dev. 7, 364-370; Modrich and Lahue, 1996, Annual Rev. Biochem.65, 101-133; Umar and Kunkel, 1996, Eur. J. Biochem. 238, 297-307).Biochemical and genetic analysis indicated that eukaryotic MutS homologs(MSH) and MutL homologs (MLH, PMS) respectively, fulfil similar proteinfunctions as their bacterial counterparts. Their relative abundance,however, could reflect different mismatch specificity and/orspecialisation for different tissues or organelles or developmentalprocesses such as mitotic versus meiotic recombination.

[0015] To date, six different genes homologous to MutS have beenisolated in yeast (yMSH and their homologs have been found in mouse(mMSH) and human (hMSH), respectively. Encoded proteins yMSH2, yMSH3 andyMSH6 appear to be the main MutS homologs involved in MMR during mitosisand meiosis in yeast, where the complementary proteins MSH3 and MSH6alternatively associate with MSH2 to recognise different mismatchsubstrates (Masischky et al., 1996, Genes Dev. 10. 407-420). Similarprotein interactions have been demonstrated for the human homologshMSH2, hMSH3 and hMSH6 (Acharya et al., 1996, PNAS 93, 13629-13634).

[0016] MutL homologs (MLH and PMS), recently reviewed by Modrich andLahue (1996, Annual Rev. Biochem. 65. 101-133) have so far been found inyeast (yMLH1 and yPMS1), mouse (mPMS2) and human (hMLH1 hPMS1 andhPMS2). The hPMS2 is a member of a family of at least 7 genes (Horii etal. 1994, Biochem. Biophys. Res. Commun. 204. 1257-1264) and its geneproduct is most closely related to yPMS1. Prolla et al. (1994, Science265, 1091-1093) presented evidence for yPMS1 and yMLH1 to physicallyassociate with each other and, together, to interact with the MutShomolog yMSH2 to form a ternary complex involved in mismatch substratebinding.

[0017] However, while medical interest in mismatch repair has promptedextensive research on MMR in bacteria, yeast and mammals, MMR genes havenot been isolated from higher plants prior to the present invention andno attempts to adjust the plant MMR to plant breeding needs have beenreported.

SUMMARY OF THE INVENTION

[0018] According to a first embodiment of the invention, there isprovided an isolated and purified DNA molecule comprising apolynucleotide sequence encoding a polypeptide functionally involved inthe DNA mismatch repair system of a plant. In one form of thisembodiment, the invention provides an isolated and purified DNA moleculecomprising a polynucleotide sequence encoding a polypeptide which ishomologous to a mismatch repair polypeptide of a yeast or of a human.More particularly, the invention provides polynucleotide sequencesencoding polypeptides which are homologous to the mismatch repairpolypeptides MSH3 and MSH6 of Saccharomyces cerevisiae. Still moreparticularly, the invention provides the coding sequences of the genesAtMSH3 and AtMSH6 of Arabidopsis thaliana, as defined hereinbelow, andpolynucleotide sequences encoding polypeptides which are homologous topolypeptides encoded by AtMSH3 and AtMSH6.

[0019] According to a second embodiment of the invention, there isprovided an isolated and purified polypeptide functionally involved inthe DNA mismatch repair system of a plant, for example a polypeptidewhich is homologous to a mismatch repair polypeptide of a yeast or of ahuman such as a polypeptide encoded by the genes AtMSH3 or AtMSH6 ofArabidopsis thaliana, as defined hereinbelow.

[0020] According to a third embodiment of the invention, there isprovided an isolated and purified DNA molecule comprising apolynucleotide sequence selected from the group consisting of (i) asequence encoding a polynucleotide which is capable of interfering withthe expression of a plant polynucleotide sequence encoding a polypeptidewhich is homologous to a mismatch repair polypeptide of a yeast or of ahuman and thereby disabling said plant polynucleotide sequence; and (ii)a sequence encoding a polypeptide capable of disrupting the DNA mismatchrepair system of a plant.

[0021] According to a fourth embodiment of the invention there isprovided a chimeric gene comprising a DNA sequence selected from thegroup consisting of (i) a sequence encoding a polynucleotide which iscapable of interfering with the expression of a plant polynucleotidesequence encoding a polypeptide which is homologous to a mismatch repairpolypeptide of a yeast or of a human and thereby disabling said plantpolynucleotide sequence, and (ii) a sequence encoding a polypeptidecapable of disrupting the DNA mismatch repair system of a plant:together with at least one regulation element capable of functioning ina plant cell. Examples of such regulation elements include constitutive,inducible, tissue type specific and cell type specific promoters such as35S. NOS, PR1a, AoPR1 and DMC1. Typically, a chimeric gene of the fourthembodiment will also include at least one terminator sequence, moretypically exactly one terminator sequence.

[0022] In the third and fourth embodiments, said interference, by saidpolynucleotide sequence, with the expression of a plant polynucleotidesequence encoding a polypeptide which is homologous to a mismatch repairpeptide of a yeast or a human typically occurs by hybridisation or byco-suppression.

[0023] According to a fifth embodiment of the invention there isprovided a plasmid or vector comprising a chimeric gene of the fourthembodiment. A vector of the fifth embodiment may be, for example, aviral vector or a bacterial vector.

[0024] According to a sixth embodiment of the invention, there isprovided a plant cell stably transformed, transfected or electroporatedwith a plasmid or vector of the fifth embodiment.

[0025] According to seventh embodiment of the invention, there isprovided a plant comprising a cell of the sixth embodiment.

[0026] According to an eighth embodiment of the invention, there isprovided a process for at least partially inactivating a DNA mismatchrepair system of a plant cell, comprising transforming or transfectingsaid plant cell with a DNA sequence of the third embodiment or achimeric gene of the fourth embodiment or a plasmid or vector of thefifth embodiment, and causing said DNA sequence to express saidpolynucleotide or said polypeptide.

[0027] According to a ninth embodiment of the invention, there isprovided a process for increasing genetic variation in a plantcomprising obtaining a hybrid plant from a first plant and a secondplant, or cells thereof, said first and second plants being geneticallydifferent; altering the mismatch repair system in said hybrid plant;permitting said hybrid plant to self-fertilise and produce offspringplants; and screening said offspring plants for plants in whichhomeologous recombination has occurred. For example, homeologousrecombination may be evidenced by new genetic linkage of a desiredcharacteristic trait or of a gene which contributes to a desiredcharacteristic trait.

[0028] According to a tenth embodiment of the invention there isprovided a process for obtaining a plant having a desiredcharacteristic, comprising altering the mismatch repair system in aplant, cell or plurality of cells of a plant which does not have saiddesired characteristic, permitting mutations to persist in said cells toproduce mutated plant cells, deriving plants from said mutated plantcells, and screening said plants for a plant having said desiredcharacteristic.

[0029] In a preferred form of the ninth and tenth embodiments of theinvention, the step of altering the mismatch repair system comprisesintroducing into said hybrid plant, plant, cell or cells a chimeric geneof the fourth embodiment and permitting the chimeric gene to express apolynucleotide which is capable of interfering with the expression of aplant polynucleotide sequence in a mismatch repair gene of the hybridplant, plant, cell or cells, or a polypeptide capable of disrupting theDNA mismatch repair system of the hybrid plant or cells.

[0030] In other embodiments, the invention provides (a) anoligonucleotide capable of hybridising at 45° C. under standard PCRconditions to a DNA molecule of the first embodiment; (b) anoligonucleotide capable of hybridising at 45° C. under standard PCRconditions to the DNA of SEQ ID NO: 18 and (c) an oligonucleotidecapable of hybridising at 45° C. under standard PCR conditions to theDNA of SEQ ID NO:30; with the proviso that the oligonucleotide of (a),(b) and (c) is other than SEQ ID NO:1 or SEQ ID NO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 provides a diagrammatic representation of the primersequences used to isolate AtMSH3.

[0032]FIG. 2 is a plasmid map of clone 52, showing restriction enzymecleavage sites in the 5′ half of the full-length cDNA for AtMSH3.

[0033]FIG. 3 is a plasmid map of clone 13, showing restriction enzymecleavage sites in the 3′ half of the full-length cDNA for AtMSH3.

[0034]FIG. 4 is a sequence listing of the coding sequence of AtMSH3,together with a deduced sequence of the encoded polypeptide.

[0035]FIG. 5 is a protein alignment of yeast (Saccharomyces cerevisiae)and Arabidopsis thaliana MSH3 protein.

[0036]FIG. 6 provides a diagrammatic representation of the primersequences used to isolate AtMSH6.

[0037] FIG. 7 is a plasmid map of clone 43, showing restriction enzymecleavage sites in the 5′ half of the full-length cDNA for AtMSH6.

[0038]FIG. 8 is a plasmid map of clone 62, showing restriction enzymecleavage sites in the 3′ half of the full-length cDNA for AtMSH6.

[0039]FIG. 9 is a sequence listing of the coding sequence of AtMSH6,together with a deduced sequence of the encoded polypeptide.

[0040]FIG. 10 is a protein alignment of yeast (Saccharomyces cerevisiae)and Arabidopsis thaliana MSH6 protein.

[0041]FIG. 11 is a genomic sequence listing of AtMSH6.

[0042]FIG. 12 is a plasmid map of plasmid pPF13.

[0043]FIG. 13 is a plasmid map of plasmid pPF14.

[0044]FIG. 14 is a plasmid map of plasmid pCW 186.

[0045]FIG. 15 is a plasmid map of plasmid pCW187.

[0046]FIG. 16 is a plasmid map of plasmid pPF66.

[0047]FIG. 17 is a plasmid map of plasmid pPF57.

[0048]FIG. 18 is a diagrammatic representation of an antisense geneconstruction for use in homeologous meiotic recombination.

[0049]FIG. 19 is a plasmid map of plasmid p3243.

DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention is based on the inventors' discovery thatthere exist in higher plants genes which are homologous to MMR genes inE. coli, and to MMR genes in yeasts and humans.

[0051] Thus, the inventors have identified genes, herein designatedAtMSH3 and AtMSH6, of the plant Arabidopsis thaliana which encode theproteins AtMSH3 and AtMSH6. These plant proteins are homologous to yMSH3and yMSH6, respectively. The present inventors have isolated cDNAsencoding the proteins AtMSH3 and AtMSH6 and have isolated the completegene encoding AtMSH6. Given the teaching herein, other genes (forexample AtMSH2, and genes of other plants) may be obtained which areinvolved in DNA mismatch repair in plants, including other genes whichencode polypeptides homologous to MMR proteins of yeasts or humans, suchas genes which encode polypeptides homologous to yeast MSH2. MLH1 orPMS2, or to human MLH1. PMS1 or PMS2. For example, given the teachingherein, genes of members of the Brassicaceae family or of otherunrelated families, for example the Poaceae, the Solanaceae, theAsteraceae, the Malvaceae, the Fabaceae, the Linaceae, the Canabinaceae,the Dauaceae and the Cucurbitaceae family, and which encode polypeptideshomologous to MMR proteins of yeasts or humans may be obtained.

[0052] Examples of plants whose genes encoding polypeptides homologousto MMR proteins of yeasts or humans may be obtained given the teachingherein include maize, wheat, oats, barley, rice, tomato, potato,tobacco, capsicum, sunflower, lettuce, artichoke, safflower, cotton,okra, beans of many kinds including soybean, peas, melon, squash,cucumber, oilseed rape, broccoli, cauliflower, cabbage, flax, hemp, hopsand carrot.

[0053] Within the meaning of the present invention, a first polypeptideis defined as homologous to a second polypeptide if the amino acidsequence of the first polypeptide exhibits a similarity of at least 50%on the polypeptide level to the amino acid sequence of the secondpolypeptide.

[0054] A procedure which may be followed to obtain genes AtMSH3 andAtMSH6 is described in Example 1. Essentially the same technique may beapplied to obtain other mismatch repair genes of Arabidopsis thaliana,and essentially the same technique as exemplified herein may be appliedto cDNA obtained by reverse transcription of RNA from other plants.Alternatively, given the sequence information disclosed herein, otherdegenerate oligonucleotide primers, especially oligonucleotides of theinvention which are capable of hybridising at 45° C. under standard PCRconditions (such as the conditions described in Example 1 using primersUPMU and DOMU) to AtMSH3 and/or AtMSH6 may be designed and obtained foruse in isolating sequences of plant mismatch repair genes which arehomologous to AtMSH3 or AtMSH6, from other plants. Similarly,oligonucleotides of the invention which are capable of hybridising at45° C. under standard PCR conditions to plant mismatch repair genes ofplants other than Arabidopsis thaliana also fall within the scope of thepresent invention and may be utilised to obtain mismatch repair genes ofstill other plants. Typically, such oligonucleotides are capable ofhybridising at 45° C. under standard PCR conditions to a DNA moleculewhich encodes a polypeptide which is homologous to a mismatch repairpolypeptide of a yeast or a human. The temperature at whicholigonucleotides of the invention hybridise to AtMSH3 and/or AtMSH6, orto plant mismatch repair genes of plants other than Arabidopsisthaliana, or to DNA molecules which encode polypeptides which arehomologous to a mismatch repair polypeptide of a yeast or a human may behigher than 45° C., for example at least 50° C., or at least 55° C. orat least 60° C. or as high as 65° C.

[0055] The successful gene isolation disclosed herein demonstrates forthe first time the existence of MMR in higher plants and indicates thepresence of other plant MMR genes. For example, genes encoding the planthomologs of MSH1. MSH2. MSH4, MSH5, PMS1. PMS2 and MLH1 may beidentified given the teaching herein. Such genes, as well as thosespecifically described herein, separately or in combination, are usefulin manipulating the plant MMR for plant breeding purposes. Thus, forexample, the plant MMR may be altered by including in a plant cell apolynucleotide sequence as defined herein above with reference to thethird embodiment of the invention, and causing the polynucleotidesequence to express either a polynucleotide which disables a plant MMRgene, or a polypeptide which disrupts the plant's MMR system.

[0056] The DNA molecule of the third embodiment of the inventionincludes a polynucleotide sequence (herein referred to as a MMR alteringgene) which may for example encode sense, antisense or ribozymemolecules characterised by sufficient base sequence similarity orcomplementarity to the gene to be altered to permit the antisense orribozyme molecule to hybridise with the plant MMR gene in vivo or topermit the sense molecule to participate in co-suppression.Alternatively, the MMR altering gene may encode a protein or proteinswhich interfere with the activity of a plant MMR protein and thusdisrupt the plant's MMR system. For example, such encoded proteins maybe antibodies or other proteins capable of interfering with MMR proteinfunction, such as by complexing with a protein functionally involved inplant MMR thereby disrupting the MMR of the plant. An example of such aprotein is the MSH3 protein of Arabidopsis thaliana described herein ora protein of another plant which is homologous to the MSH3 protein of A.thaliana. For instance, overexpression of MSH3 in a plant cell causesMSH2 present in the cell to be substantially completely complexed,disrupting the mismatch repair mechanism or mechanisms in the cell whichare functionally dependent on the presence of a complex of MSH2 withMSH6. Similarly, mismatch repair mechanisms which depend on the presenceof a complex of MSH2 and MSH3 may be disrupted by the overexpression ofMSH6.

[0057] A chimeric gene of the fourth embodiment, incorporating a MMRaltering gene, may be prepared by methods which are known in the art.Similarly, the MMR altering gene may be introduced into a plant cell,regenerating tissue or whole plant by techniques known in the art asbeing suitable for plant transformation, or by crossing. Knowntransformation techniques include Agrobacterium tumefaciens or A.rhizogenes mediated gene transfer, ballistic and chemical methods, andelectroporation of protoplasts.

[0058] The MMR altering gene or genes are typically expressed fromsuitable promoters. Suitable promoters may direct constitutiveexpression, such as the 35S or the NOS promoter. Usually, however, thepromoter will direct either inducible or tissue specific (e.g. callus;embryonic tissue; etc.), cell type specific (e.g. protoplasts;meiocytes; etc.) or developmental (e.g. embryo) expression of thealtering gene or genes, in order for the MMR system to function intissue types or cell types, or at developmental stages of the plant, inwhich it is not desirable for the MMR system to be altered. Using suchpromoters, therefore, the activity of a MMR altering gene may be limitedto a specific stage during plant development or it may be altered bycontrolling conditions external to the plant, and the deleteriouseffects of a permanently disabled or altered DNA mismatch repair systemin a plant may be avoided. Examples of suitable promoters which are notconstitutive are known in the art and include inducible promoters suchas PR1a (reviewed by Gatz, 1997, Annual Rev. Plant Phys. Plant Mol.Biol. 48: 89), tissue specific promoters such as AoPR1 (Sabahattin etal., 1993, Biotechnology 11: 218), and cell-type specific promoters suchas DMC1.

[0059] A chimeric gene in accordance with the invention may further bephysically linked to one or more selection markers such as genes whichconfer phenotypic traits such as herbicide resistance, antibioticresistance or disease resistance, or which confer some otherrecognisable trait such as male sterility, male fertility, grain size,colour, growth rate, flowering time, ripening time, etc.

[0060] The process of the tenth embodiment of the invention provides,for example, a process for generating intraspecies genetic variation byaltering the mismatch repair system in a plant cell, in regeneratingplant tissue or in a whole plant. The plant cell, regenerating tissue orwhole plant includes and expresses one or more MMR altering genes whichare capable of altering mismatch repair in the plant cell, regeneratingtissue or whole plant. Alteration of MMR may be achieved, for example,by inactivating the genes encoding plant MSH3 and/or plant MSH6. It ispreferred to inactivate the plant MSH3 and MSH6 encoding genes at thesame time and in the same plant cell, regenerating tissue or wholeplant. Typically in this preferred form of the invention inactivation ofeither plant MSH3 or MSH6 alone is insufficient to substantially alterthe plant's mismatch repair system and only when both MSH3 and MSH6 areinactivated simultaneously is the plant's mismatch repair systemsufficiently altered to prevent the MMR system from recognising basepair mismatches, base insertions or deletions as a result of DNAreplication errors, DNA damage, or oligonucleotide induced site-specificmutagenesis. However, in some applications of the invention,inactivation of only one gene may also be used to cause genomicinstability or increase the efficiency of site-specific mutagenesis.

[0061] If desired, the MMR altering gene or genes may later be renderednon-functional or ineffective, or may be removed from the genome of theplant cell, regenerating tissue or whole plant in order to restoremismatch repair in the plant cell, regenerating tissue or whole plant.The MMR altering gene or genes may be inactivated by means of known geneinactivation tools, such as ribozymes, or may be removed from the genomeusing gene elimination systems known in the art, such as CRE/LOX. It ispreferred to render two genes, whose gene products combine toincapacitate MMR, ineffective by separating the altering genes throughsegregation. Therefore, in a preferred embodiment of the invention afirst plant cell or plant is generated in which only plant MSH3 isincapacitated, and a second plant cell or plant is generated in whichonly plant MSH6 is incapacitated. The combination of both genomes, forexample by crossing, then produces significant MMR deficiency in thosecells or plants which have inherited both altering genes. If thealtering genes are expressed from unlinked loci, gene segregationrestores MMR activity in the progeny of the cells or plants.

[0062] In a process of the ninth embodiment of this invention,homeologous recombination is enhanced between different genomes,chromosomes or genes in plant cells or plants by altering MMR in saidplant cells or plants. Such genomes, chromosomes or genes arecharacterised in that they originate from different plant families,genera, species, subspecies, plant varieties or lines. Hybrid plantcells or hybrid plants may be produced by crossing, by cell fusion or byother techniques known in the art. These plant cells or plants arefurther characterised by expressing one or more genes that are capableof altering mismatch repair in the plant cell or plants.

[0063] In the process of the ninth embodiment, the homeologousrecombination is typically for the purpose of introducing a desiredcharacteristic in the hybrid plant. In this typical application of theprocess of the ninth embodiment, and in the process of the tenthembodiment the desired characteristic may be any characteristic which isof value to the plant breeder. Examples of such characteristics are wellknown in the art and include altered composition or quality of leaf orseed derived storage products (e.g. oil, starch, protein), alteredcomposition or quality of cell walls (e.g. decrease in lignin content),altered growth rate, prolonged flowering, increased plant yield or grainyield, altered plant morphology, resistance to pathogens, tolerance toor improved performance under environmental stresses of various kinds,etc.

[0064] In a preferred form of the tenth embodiment, an MMR altering geneis co-introduced along with the homeologous genome, chromosome or geneof another plant cell or plant into an MMR proficient plant cell or MMRproficient plant to produce a hybrid plant cell or hybrid plant in whichhomeologous recombination can occur. Suitably, the MMR proficient plantcell or MMR proficient plant may also include an MMR altering gene. Forexample a gene capable of inactivating plant MSH3 may be co-introducedalong with the homeologous genome, chromosome or gene of another plantcell or plant into an MMR proficient plant cell or MMR proficient plantin which MSH6 is inactivated. A resultant hybrid plant in whichhomeologous recombination occurs will include both the MSH3 and MSH6altering genes and its MMR system will therefore be inactivated.

[0065] In this form of the invention, if hybrid plants are to beproduced by crossing, the MMR altering gene preferably originates fromthe male parent, thus ensuring that the MMR altering gene is alwaysintroduced and is not present in the recipient cell. That is, the MMR ofthe recipient cell, prior to introduction of the MMR altering gene, istypically proficient. Alternatively, if an MMR altering gene is presentin a recipient cell it may be ineffective or inefficient on its own, orit may be linked to an inducible or tissue specific or cell typespecific promoter which only renders the MMR altering gene active underlimited conditions.

[0066] Thus, in a preferred form of the process of the ninth embodiment,the MMR system of the hybrid plant is initially unaltered. In this formof the process, the step of altering the mismatch repair system maycomprise introducing into the hybrid plant, or cells thereof, a MMRaltering gene, such as by Agrobacterium tumefaciens or A. rhizogenesmediated gene transfer, ballistic and chemical methods, andelectroporation of protoplasts.

[0067] The MMR altering gene or genes are typically expressed fromsuitable promoters, as described above. Preferably, the promoter istranscriptionally active in mitotically and meiotically active tissueand/or cells to ensure MMR alteration after chromosome pairing atmitosis and meiosis, respectively. The preferred timing for MMRalteration is at meiosis, because recombinant genomes, chromosomes orgenes are directly transmitted to the progeny. A suitable meiocytespecific promoter is for example the DMC1 promoter from Arabidopsisthaliana ssp. Ler. (Klimyuk and Jones, 1997, Plant J. 11, 1-14).However, mitotic homeologous recombination is also a desirable outcomeas somatic recombination events can be transmitted to offspring due tothe totipotency of plant cells and the lack of predetermined germ cellsin plants.

[0068] If desired, the MMR altering gene or genes may later be renderednon-functional or ineffective, or may be removed from the hybrid plantor hybrid plant cells, in order to restore mismatch repair in the hybridplant or hybrid plant cells. The MMR altering gene or genes may beinactivated by means of known gene inactivation tools as describedherein above.

EXAMPLES Example 1 Cloning of the AtMSH3 and AtMSH6 Coding Sequences

[0069] Isolation of Partial AtMSH3 and AtMSH6 Consensus Sequences

[0070] Degenerate oligonucleotides UPMU (SEQ ID NO:1) and DOMU (SEQ IDNO.2) UPMU CTGGATCCACIGGICCIAA(C/T)ATG DOMUCTGGATCC(A/G)TA(A/G)TGIGTI(A/G)C(A/G)AA

[0071] were used to isolate AtMSH3 and AtMSH6 sequences by PCRamplification.

[0072] Primers UPMU and DOMU correspond to conserved amino acidsequences of the proteins MutS (E. coli and S. typhimurium), HexA (S.pneumoniae). Rep1 (mouse) and Duc1 (human). The conserved regions towhich they are targeted are TGPNM for UPMU (amino acid positions 852-856for AtMSH6 and 816-820 for AtMSH3) FATHY or FVTHY for DOMU (amino acidpositions 964-968 for AtMSH6 and 928-932 for AtMSH3, respectively.)These primers have been used to isolate MSH2 and MSH1 from yeast (Reenanand Kolodner. Genetics 132: 963-97 (1992)) and MSH2 from Xenopus andmouse (Varlet et al. Nuc. Acids Res. 22:5723-5728 (1994)).

[0073] Template single strand cDNA was produced by reverse transcriptionof 2 μg total RNA from a cell suspension culture of Arabidopsis thalianaecotype Columbia (Axelos et at. 1989, Mol. Gen. Genetics 219: 106-112).The PCR reaction was performed under the following conditions in a finalvolume of 100 μl: 0.2 mM dNTP, 1 μM each primer, 1×PCR buffer, 1 u TaqDNA polymerase (Appligene) in the presence of template cDNA. PCRparameters were 5 minutes at 94° C., followed by 30 cycles of 40 secondsat 95° C., 90 seconds at 45° C., 1 minute at 72° C. The amplificationproducts were cloned into pGEM-T vector (Promega) and sequenced. Twodifferent clones were isolated, S5 (350 bp) was homologous to MSH3, S8(327 bp) was homologous to MSH6. Complete cDNA sequences were thenisolated according to the Marathon cDNA amplification kit procedure(Clontech). In summary, this procedure involves producing doublestranded cDNA by reverse transcription of 2 μg polyA+ RNA from the cellsuspension culture of Arabidopsis. Adaptors are ligated on each side ofthe cDNA. The ligated cDNA is used as a template for 5′ and 3′ RACE PCRreactions in the presence of primers that are specific for the adaptoron one side (AP1 and AP2), and specific for the targeted gene on theother side. A 5′ and a 3′ fragment that overlap are thus produced foreach gene. The complete gene coding sequence can be reconstituted takingadvantage of a unique restriction site, if available, in the overlappingregion. Specific details of this procedure as it was used to isolateAtMSH3 and AtMSH6 coding regions, are as follows.

[0074] Isolation of AtMSH3 Complete Coding Sequence

[0075] From the sequence of clone S5, primer 636 (SEQ ID NO:3) wasdesigned: 636 TGCTAGTGCCTCTTGCAAGCTCAT.

[0076] Primer AP1 (SEQ ID NO:4) is complementary to a portion of anadaptor sequence which had been ligated to the 5′ and 3′ ends ofArabidopsis cDNA: AP1 CCATCCTAATACGACTCACTATAGGGC.

[0077] PCR performed on the ligated cDNA with primers 636 and AP1 forthe 5′ RACE PCR was followed by a second round of amplification with thenested primers AP2 (SEQ ID NO:5) and S525 (SEQ ID NO:6) AP2ACTCACTATAGGGCTCGAGCGGC S525 AGGTTCTGATTATGTGTGACGCTTTACTTA

[0078] (the latter was also designed to correspond to a part of thesequence of clone S5) and produced a 2720 bp DNA fragment. FIG. 1provides a diagrammatic representation of the primer sequences used toisolate AtMSH3. Another primer (S51, SEQ ID NO:7) S51GGATCGGGTACTGGGTTTTGAGTGTGAGG

[0079] was designed closer to the 5′ border and permitted thedetermination of 99 bp upstream to the ATG initiation codon. For the 3′RACE PCR, a first PCR reaction was performed with primers API and 635(SEQ ID NO:8). 635 GCACGTGCTTGATGGTGTTTTCAC

[0080] followed by a second round of amplification, using the nestedprimers AP2 and S523 (SEQ ID NO:9) S523 TCAGACAGTATCCAGCATGGCAGAAGTA

[0081] which produced a DNA fragment of 890 bp. Both DNA fragments weresubcloned into pGEM-T and sequenced. Since PCR amplification using theExpand Long Template PCR System (Boehringer-Mannheim) produced errors inthe sequence, new oligonucleotides were designed to isolate thosesequences again by PCR, but with the high fidelity DNA polymerase Pfu.PCR with primers 1S5 (SEQ ID NO:10) and S53 (SEQ ID NO:11) 1S5ATCCCGGGATGGGCAAGCAAAAGCAGCAGACGA S53 GACAAAGAGCGAAATGAGGCCCCTTGG

[0082] amplified the 1244 bp fragment clone 52 (SEQ ID NO:12′, clonedinto pUC18SmaI). PCR with primers S52 (SEQ ID NO:13) and 2S5 (SEQ IDNO:14) 2S5 ATCCCGGGTCAAAATGAACAAGTTGGTTTTAGTC S52GCCACATCTGACTGTTCAAGCCCTCGC

[0083] amplified the 2104 bp clone 13 (SEQ ID NO:15, cloned intopUC18/SmaI). The complete coding sequence of the AtMSH3 gene wasreconstructed in pUC18 by ligating the 5′ half of AtMSH3 (clone 52) tothe 3′ half of AtMSH3 (clone 13) after digesting with BamH1 which has aunique cleavage site in the overlapping region of both clones. Thismanipulation yielded plasmid pPF26. The SmaI fragment from pPF26contains the complete AtMSH3 coding sequence. The remaining primersreferred to in FIG. 1 are as follows: S51 GGATCGGGTACTGGGTTTTGAGTGTGAGG(SEQ ID NO:16) S525 AGGTTCTGATTATGTGTGACGCTTTACTTA (SEQ ID NO:17)

[0084]FIGS. 2 and 3 provide plasmid maps of clones 52 and 13respectively, showing restriction enzyme cleavage sites. The completeAtMSH3 coding sequence (SEQ ID NO:18) is 3246 bp long and is shown inFIG. 4 together with the deduced sequence (SEQ ID NO:19) of the encodedpolypeptide. AtMSH3 is clearly homologous to the yeast and mouse MSH3genes. A sequence alignment of polypeptides encoded by AtMSH3 and thatencoded by Saccharomyces cerevisiae MSH3 is set out in FIG. 5.

[0085] Isolation of the AtMSH6 Complete Coding Sequence and GenomicSequences

[0086] The same procedure allowed isolation of the AtMSH6 cDNA. FIG. 6provides a diagrammatic representation of the primer sequences used toisolate AtMSH6. For the 5′ RACE PCR, primers 638 (SEQ ID NO:20) and API(SEQ ID NO:4) 638 TCTCTACCAGGTGACGAAAAACCG

[0087] allowed the amplification of a 2889 DNA fragment. Primer S81 (SEQID NO:21) S81 CGTCGCCTTTAGCATCCCCTTCCTTCAC

[0088] helped define the 142 bp upstream to the ATG initiation codon. Onthe 3′ side. RACE PCR was initially performed with primers S82′3 (SEQ IDNO:22) and AP1 (SEQ ID NO:4), S823 GCTTGGCGCATCTAATAGAATCATGACAGG

[0089] and then with the nested primers 637 (SEQ ID NO:23) and AP2 (SEQID NO:5). 637 GACAGCGTCAGTTCTTCAGAATGC

[0090] to produce a 774 bp DNA fragment. As for AtMSH3, those fragmentswere cloned and sequenced. Re-isolation of the DNA sequence using thehigh fidelity Pfu polymerase and newly designed primers 1S8 (SEQ IDNO:24) and S83 (SEQ ID NO:25) (for the 5′ side) led to a 2182 bp DNAfragment identified as clone 43 (SEQ ID NO:26, cloned in pUC18/SmaI),and a 1379 bp clone identified as clone 62 (SEQ ID NO:27, also cloned inpUC18/SmaI). 1S8 ATCCCGGGATGCAGCGCCAGAGATCGATTTTGT 2S8ATCCCGGGTTATTTGGGAACACAGTAAGAGGATT (SEQ ID NO: 28) S82GCGTTCGATCATCAGCCTCTGTGTTGC (SEQ ID NO: 29) S83CGCTATCTATGGCTGCTTCGAATTGAG

[0091]FIGS. 7 and 8 provide plasmid maps of clones 43 and 62respectively, showing restriction enzyme cleavage sites. Clones 43 and62 were digested by the Xmn1 restriction enzyme for which a unique siteis present in their overlapping region and then ligated. The completeAtMSH6 coding sequence (SEQ ID NO:30) is 3330 bp long and is shown inFIG. 9 together with the deduced sequence (SEQ ID NO:31) of the encodedpolypeptide. AtMSH6 is clearly homologous to the yeast and mouseMSH6genes. A sequence alignment of polypeptides encoded by AtMSH6 andthat encoded by Saccharomyces cerevisiae MSH6 is set out in FIG. 10.

[0092] An AtMSH6 genomic sequence was also isolated from a genomic DNAlibrary constituted after partial Sau3AI digestion of DNA from theArabidopsis cell suspension. 8062 bp were sequenced that covered theAtMSH6 gene and show colinearity with the cDNA. 16 introns are foundscattered along the gene. The complete genomic sequence (SEQ ID NO:98)is shown in FIG. 11.

Example 2 A Measure of Somatic Variation in MMR Deficient Plants

[0093] Constructs

[0094] Constructs with antisense AtMSH3 or antisense AtMSH6 or bothAtMSH3/AtMSH6 under the control of a single 35S promoter have beeninserted into the binary vector pPZP121 (Hajdukiewicz et al., Plant Mol.Biol. 23, 793-799) between the right and left borders of the T-DNA. ThepPZP121 plasmid confers chloramphenicol resistance to Escherichia colior Agrobacterium tumefaciens bacteria. The aacC1 gene is carried by theT-DNA and allows selection of transformed plant cells on gentamycin(Hajdukiewicz et al., Plant Mol. Biol. 25, 989-994). For the purpose ofexpressing antisense constructs, a 35S promoter/terminator cassette witha polylinker was introduced into pPZP121. The 3′ ends of the consideredgenes have been chosen since this region seems more efficient forantisense inhibition. For AtMSH3 this corresponds to clone 13 (2104 bp),for AtMSH6 this corresponds to clone 62 (1379 bp). Clone 13 comprises2104 bp of the 3′ region that were cut off the pUC18 vector by SalI/Sstlrestriction, blunted with T4 DNA polymerase and ligated into the T4 DNApolymerase blunted BamHI site of pPZP121/35S, creating clone pPF13. Thesame procedure was followed for the 3′ region of AtMSH6 clone 62 (1379bp) thus creating plasmid pPF14. For the double constructs, the 3′region (from clone 62) of AtMSH6 was introduced ahead of the AtMSH3region into pPF13 creating pCW186 and reciprocally, the 3′ region ofAtMSH3 (from clone 13) was introduced ahead of AtMSH6 into pPF14,creating pCW187.

[0095] These constructs were introduced into the Arabidopsis cells (asdescribed below) of wildtype Columbia and of the Columbia tester line.

[0096] An alternative strategy to antisense inhibition of AtMSH6 comesfrom experiments of Marra et al. (1998. Proc. Natl. Acad. Sci USA 95.8568-8573) who show that overexpression of functional MSH3 results indepletion of MSH6 protein in human cells. This depletion may generate amismatch repair mutant phenotype.

[0097] For the purpose of overexpressing functional AtMSH3 protein inplant cells, the complete MSH3 coding region was excised from pPF26(example 1) by digestion with SmaI, and was inserted into the SmaI siteof pCW164. The resulting construct was named pPF66. It contains acomplete AtMSH3 gene under the control of the 35S promoter inside theleft (LB) and right (RB) border of the T-DNA. This T-DNA also containsthe hpt2 gene for gentamycin selection. Plasmid pPF66 was introducedinto Arabidopsis cells as described below. One cell clone was selectedwhich clearly overexpressed the AtMSH3 gene as shown by Northernanalysis. FIGS. 12-16 provide plasmid maps of plasmids pPF13, pPF14,pCW186, pCW187 and pPF66, respectively.

[0098] Construction of Tester Construct

[0099] For the purpose of Forward Mutagenesis Assays, a tester constructwas built containing the coding regions for nptII, codA, uidA. All threegenes are driven by the 35S promoter and are terminated by the 35Sterminator. This construct was obtained by introducing an EcoRI fragmentencoding the codA cassette (2.5 kb) and a HindIII fragment encoding theuidA (GUS) cassette (2.4 kb) into the pPZP111 vector (Hajdukiewicz etal., 1994, Plant Mol Biol 23: 793-799) which already contained the nptIIexpression cassette. This new plasmid was named pPF57. NptII is used toselect for transformed plant cells. GUS is used to analyse the degree ofgene silencing in the construct (i.e. to identify cell lines in whichthe transgenes are expressed), and codA is used as a marker for forwardmutagenesis (described below).

[0100] The plasmid map of pPF57 is provided in FIG. 17.

[0101] Plant Cell Transformation

[0102] The constructs are introduced into Agrobacterium byelectroporation. Plant cells are then transformed by co-cultivation. Asuspension culture of Arabidopsis thaliana cells that has beenestablished by Axelos et al. (1992, Plant Physiol. Biochem. 30, 1-6) maybe used. One day old freshly subcultured cells are diluted five timesinto AT medium (Gamborg B5 medium. 30 g/l sucrose, 200 μg/l NAA). 10 μlof saturated Agrobacterium containing the transforming T-DNA constructsare added to 10 ml diluted cells in a 100 ml erlenmeyer. Theco-cultivation is agitated slowly (80 rpm) for 2 days. The cells arethen washed 3 times into AT medium and finally resuspended in the sameinitial volume (10 ml). The culture is agitated for 3 days to allowexpression before plating on selection plates (AT/BactoAgar 8g/l+gentamycin 50 μg/ml). Transformed individual calli are isolated 3weeks later.

[0103] Tester Strain

[0104] The tester construct on plasmid pPF57 was introduced intoArabidopsis cells of wildtype Columbia using the transformation protocoldescribed above. Among 10 candidate transformants, one cell clone wasshown (by Southern analysis) to have a unique T-DNA insertion. All threegenes were shown to be functional in this cell line as indicated byresistance to kanamycin, blue staining in the presence of X-Glu (GUS),and sensitivity to 5-fluoro-cytosine (codA).

[0105] MMR altering genes (described above) were then introducedindividually into the tester line and transformed cells are used foranalysis of both Microsatellite Instability and Forward Mutagenesis.

[0106] Microsatellite Analysis

[0107] Microsatellites have been described in Arabidopsis (Bell andEcker, 1994, Genomics 19, 137-144). The present Example is based on astudy of instability of microsatellites that are polymorphic amongstdifferent ecotypes. DNA is extracted from the transformed calli.Specific primers have been defined that are used to amplify themicrosatellite sequence. One of the two primers is previously P³²labelled by T4 kinase. In case of a polymorphic variation, new PCRproducts appear that do not follow the expected pattern of migration ona polyacrylamide gel. This is a commonly observed feature for MMRdeficient cells in yeast or mammalian cells.

[0108] In particular, the present Example describes a study onmicrosatellites ca72 (CA₁₈), ngaI72 (GA₂₉), and ATHGENEA (A₃₉), chosenbecause they belong to the types predominantly affected in humanmismatch repair deficient tumors. The size of these microsatellites isnot conserved from one Arabidopsis ecotype to the other.

[0109] Arabidopsis cells which are transformed with, an MMR alteringgene (above) and control cells not expressing the MMR altering gene areallowed to form calli. DNA is rapidly extracted from the calli and isanalysed for microsatellite instability as described in detail by Belland Ecker 1994, Genomics 19, 137-144. In summary, the relevantmicrosatellite is amplified by PCR using P32 labelled primers. The PCRproducts are separated on a DNA sequencing gel for size determination.Size differences between microsatellites from transformed and controlcells not expressing the MMR altering gene in question indicatemicrosatellite instability as a result of MMR alteration.

[0110] The sequences of primers used for PCR amplification ofmicrosatellites ca72 and ngaI72 are included in Table 1. PCRamplification of microsatellite ATHGENEA made use of a forward primercontaining the sequence ACCATGCATAGCTTAAACTTCTTG (SEQ ID NO: 32)

[0111] and of a reverse primer containing the sequenceACATAACCACAAATAGGGGTGC. (SEQ ID NO: 33)

[0112] The amplification for microsatellite ca72 revealed in Columbiacontrol cells (with respect to the MMR altering gene) a 248 bp long PCRfragment instead of the published length of 124 bp. DNA sequencingverified this fragment as a CA₁₈ microsatellite.

[0113] Forward Mutagenesis Assay

[0114] Tester cells transformed with antisense AtMSH3 or antisenseAtMSH6 or both AtMSH3/AtMSH6 are analysed for the stability of the codAgene. The functional codA gene confers to sensitivity to5-fluoro-cytosine (5FC), whereas a gene inactivating mutation in codAwill confer resistance to 5FC. The frequency of resistant cells istherefore a good indicator of somatic variation as a direct result ofMMR alteration. Variants resistant to 5FC are first analysed for GUSactivity. If GUS is inactive, 5FC resistance is assumed to be due togene silencing (all three genes are under the 35S promoter). If GUS isactive, 5FC resistance is assumed to be due to forward mutations thathave inactivated codA. PCR is then performed on the putative codA mutantgenes which is then sequenced to confirm the presence of forwardmutations in codA.

[0115] Besides codA, other marker genes may also be used for the ForwardMutagenesis Assay such as the ALS gene (conferring sensitivity to valineor to sulfonylurea; Hervieu and Vaucheret, 1996, Mol. Gen. Genet. 251220-224: Mazur et al. 1987, Plant Physiol. 85 1110-1117).

Example 3 Homeologous Meiotic Recombination in Arabidopsis Thaliana

[0116] A. Construction of a Meiocyte Specific Gene Expression CassetteComprising the DMC1 Promoter and the NOS Terminator

[0117] (i) The DMC1 promoter may be used as published by Klimyuk andJones, 1997, Plant J. 11.1-14). To obtain a more convenient alternativefor gene cloning, a 3.3 Kb long subfragment of the DMC1 promoter wasobtained by PCR from genomic DNA of Arabidopsis thaliana (ssp. Landsbergerecta “Ler”).

[0118] The PCR was done in three rounds:

[0119] Round One: A 3.7 Kb long product was obtained using the forwardprimer DMCIN-A comprising the sequence GAAGCGATATTGTTCGTG (SEQ ID NO:34)

[0120] and the reverse primer DMCIN-B comprising the sequenceAGATTGCGAGAACATTCC. (SEQ ID NO: 35)

[0121] The weak amplification product was then used as template forround two and three.

[0122] Round Two: A 3.1 Kb long product comprising the promoter and the5′ untranslated leader was obtained using forward primer DMCIN-1, whichcontained the sequence acgcgtcgacTCAGCTATGAGATTACTCGTG (SEQ ID NO :36)

[0123] and introduced a SalI cloning site at the 5′ end of the promoterfragment, and reverse primer DMCIN-2 which contained the sequencegctctagaTTTCTCGCTCTAAGACTCTCT (SEQ ID NO:37)

[0124] and introduced a XbaI site at the 3′ end of the PCR fragment.

[0125] Round Three: A 0.2 Kb long product comprising the firstexon/intron of the DMC1 promoter was obtained using forward primerDMCIN-3, which contained the sequence gctctagaGCTTCTCTTAAGTAAGTGATTGAT(SEQ ID NO:38)

[0126] and introduced a XbaI site at the 5′ end of the PCR fragment, andreverse primer DMCIN-4, containing the sequencetcccccgggctcgagagatctccatggTTTCTTCAGCTCTATGAATCC (SEQ ID NO:39)

[0127] and introduced at the 3′ end of the PCR product restriction sitesfor NcoI. BglII, XhoI and SmaI.

[0128] The products obtained in round Two and Three were digested withXbaI and subsequently ligated to reconstitute a 3.3 Kb long DMC1promoter from which the first two in-frame ATG start codons werereplaced with a unique restriction site for XbaI. This promoter can becloned between the restriction sites for SalI and SmaI of p3264, whichcontains the SacI-EcoRI NOS terminator in pBIN19, to yield the entireexpression cassette in pBIN19. This cassette contains the followingcloning sites: NcoI, BglII, XhoI. SmaI and (already present on p3264)KpnI and SacI.

[0129] (ii) Another strategy yielded the following convenient DMC1promoter. A 1.8 kb DNA fragment comprising the 3′ terminal part of themeiocyte specific DMC1 promoter was isolated by PCR from purifiedgenomic DNA of Arabidopsis thaliana (ssp. Landsberg erecta “Ler”). Theforward PCR primer (DMC1a) contained the sequenceacgcgtcgacGAATTCGCAAGTGGGG (SEQ ID NO:40)

[0130] and introduced a SalI cloning site at the 5′ end of the promoterfragment. The reverse PCR primer (DMC1b) contained the sequencetccatggagatctcccgggtacCGATTTGCTTCGAGGG (SEQ ID NO:41)

[0131] introducing a polylinker region at the 3′ end of the promoterfragment. The PCR reaction was carried out with VENT DNA Polymerase(NEB) over 25 cycles using the following cycling protocol: 1 minute at94° C., 1 minute at 54° C., 2 minutes at 72° C.

[0132] The PCR reaction yielded a blunt ended DNA fragment which wasdigested with restriction enzyme SalI and was cloned into the cleavagesites of restriction enzymes SalI and SmaI in plasmid p2030, a pUC118derivative containing the SacI-EcoRI NOS terminator fragment of pBIN121.The cloning yielded plasmid p2031, containing the DMC1promoter-polylinker-NOS terminator expression cassette depicted in FIG.18.

[0133] B. Construction of an MSH3 Antisense Gene Under the Control ofthe DMC1 Promoter

[0134] A 2.1 kb DNA fragment encoding the carboxyterminal part of AtMSH3was removed from the polylinker of clone 13 described in Example 1 after(i) digestion with KpnI, (ii) blunting of the DNA ends generated by KpnIand (iii) digestion with BamHI. The isolated fragment was then cloned inantisense orientation downstream of the DMC1 is promoter in plasmidp2031, which had been digested with restriction enzymes SmaI and BglII.This cloning yielded plasmid p2033 (FIG. 18).

[0135] After digestion of p2033 with EcoRI, a 4.1 kb DNA fragment wasrecovered comprising the DMC1 promoter, the partial MSH3 cDNA inantisense orientation with respect to the promoter and the NOSterminator. This fragment was cloned into the EcoRI restriction site ofplant transformation vector pNOS-Hyg-SCV to yield plasmid p3242 (FIG.18).

[0136] C. Construction of a Combined MSH6/MSH3 Antisense Gene Under theControl of a Single DMC1 Promoter

[0137] A 3.1 kb fragment, encoding in antisense orientation the partialAtMSH6 and AtMSH3 sequences and the 35S terminator, was isolated frompCW186 by digestion with KpnI. The fragment was treated with Klenowenzyme to blunt both ends. It was then cloned into the SmaI site ofplasmid p3243 (a pNOS-Hyg-SCV derivative, illustrated in FIG. 19), whichhad been opened to delete the region between the SmaI sites. Clonescontaining the fragment in the antisense orientation with respect to theDMC1 promoter (described in A (ii) above) were identified by diagnosticdigestion with BamHI. The selected construct was named p3261.

[0138] Another practical way of cloning the double antisense gene is asfollows. A 1 kb DNA fragment encoding the carboxyterminal part of AtMSH6is isolated from clone 62 described in Example 1 after digestion ofclone 62 plasmid DNA with BamHI, which cleaves in the 5′ polylinkerregion flanking the partial cDNA, and with EcoRI, which cleaves withinthe cDNA. The isolated fragment is treated with Klenow enzyme to bluntboth its ends and is cloned into the recipient plasmid p2033 or p3242.For the purpose of cloning, the recipient plasmid may be cleaved witheither AvaI or NcoI and can be blunted with Klenow enzyme to produceblunt acceptor ends for fragment cloning. This cloning yields twoopposite orientations of cloned fragment DNA with respect to the DMC1promoter. These can be identified by diagnostic digestion withrestriction enzymes ScaI or XmnI in conjunction with SacI. The selectedconstruct contains the DMC1 promoter, the combined partial cDNAs forAtMSH3 and AtMSH6 (both cloned in antisense orientation with respect tothe DMC1 promoter) and the NOS terminator. If the recipient plasmid isp2033, the combined antisense gene under control the single DMC1promoter is recovered from the vector after EcoRI digestion and iscloned into the EcoRI restriction site of pNOS-Hyg-SCV.

[0139] D. Construction of a Full-length MSH3 Sense Gene Under Control ofthe DMC1 Promoter for Overexpression of Functional MSH3 Protein

[0140] Overexpression of MSH3 protein was shown in human cells (Marra etal., 1998, Proc. Natl. Acad. Sci. USA 95. 8568-8573) to complex allavailable MSH2 protein. This leaves MSH6 protein without its partner,leading to the degradation of MSH6 protein and eventually to a mismatchrepair phenotype.

[0141] This phenomenon is exploited to increase homeologous meioticrecombination in Arabidopsis as an alternative to antisense inhibitionof AtMSH6. For this purpose the full-length cDNA encoding AtMSH3 isisolated from plasmid pPF66 by digestion with SmaI and is cloned intothe SmaI site of the DMC1 expression cassettes described in A (i).

[0142] E. Selection of Recombination Markers on Homeologous Chromosomesof Arabidopsis Thaliana Subspecies Landsbere Erecta (Ler). Columbia(Col) and C24, Respectively

[0143] E (i). Visual Recombination Markers in Arabidopsis th.,Subspecies C24:

[0144] Agrobacterium mediated transformation with a T-DNA containing a35S-GUS gene, inactivated by insertion of a 35S-Ac transposable element(Finnegan et al., 1993, Plant Mol. Biol. 22, 625-633), had yielded a C24line in which the T-DNA construct was integrated into chromosome 2.Genetic and molecular analysis of this line shows that the Ac transposonhad excised from its T-DNA locus thereby restoring GUS activity, but hadre-inserted into the chromosome at a distance of 16.4 cM, where itstayed fixed (due to disablement of Ac) within the chlorina gene.Insertional inactivation of the chlorina gene caused a bleachedphenotype in those plants that were homozygous for this mutation.Because of the two linked phenotypic markers, chlorina3A:Ac and GUS,this C24 line was used in crosses to wildtype Ler for analysis ofmeiotic homeologous recombination on chromosome 2 in conjunction withmolecular recombination markers.

[0145] E (ii). Visual Recombination Markers in Arabidopsis th. Ler:

[0146] The Ler line NW1 (obtained from NASC, Nottingham, UK) containsone recessive visual marker per chromosome. i.e. an-1 on Chr.1, py-1 onChr.2, gll-1 on Chr.3, cer2-1 on Chr.4, and ms1-1 on Chr.5. This line isused in crosses to wildtype C24 which expresses an MMR altering gene foranalysis of meiotic homeologous recombination on chromosomes 1-5 inconjunction with molecular recombination markers listed in Table 1.

[0147] Other Ler lines from NASC have several visual markers in closeproximity to each other on the same chromosome. When these lines areused for hybrid production, analysis of homeologous meioticrecombination may make use entirely of visual recombination markers.Particularly suitable for crossing to C24 wildtype that is expressing aMMR altering gene are the following Ler lines:

[0148] NW22: relative markers are dis1-(4 cM)-ga4-(11 cM)-th1 onchromosome 1.

[0149] NW10: relevant markers are tz-201-(5 cM)-cer3 on chromosome 5.

[0150] NW134, relevant markers are ttg-(4 cM)-ga3 on chromosome 5.

[0151] NW24 (abi3-1) and NW64 (gl1-1). When present in the same plant onchromosome 3, abi3-1 and gl1-1 are 13 cM apart. Since this markercombination is not available from NASC, we have combined these markersby crossing line NW24 to line NW64. The Fl offspring were allowed toself-fertilise and to produce F2 seeds. F2 Plants which carry bothmarkers as homozygous traits on the same chromosome can be identifiedfirstly, by germinating F2 seeds on germination medium containingselective concentrations of abscisic acid, and subsequently, byidentifying among the abscisic acid resistant plants those individualswhich show the glabra phenotype.

[0152] E (iii) Molecular Recombination Markers in Col. Ler and C24:

[0153] The genome of Arabidopsis thaliana is interspersed with uniquebase sequences arranged as simple tandem repeats. Allelic repeats canvary in length between different Arabidopsis subspecies and whenamplified by PCR yield diagnostic DNA products of different length namedSimple Sequence Length Polymorphisms (SSLPs). Many SSLPs have beengenetically mapped and have been assigned to unique chromosome locationson the recombinant inbred map (Bell and Ecker, 1994, Genomics 19,137-144; Lister and Deans lines, Weeds World 4i, May 1997).

[0154] In Table 1 are listed 28 mapped and established SSLPs between Lerand Col. A number of PCR primer pairs are described herein (SEQ ID NO:42to SEQ ID NO:97) which also yielded SSLPs between C24 and Ler (19 SSLPs)or between C24 and Col (25 SSLPs), respectively. Polymorphic SSLPs canbe used as molecular markers in the analysis of homeologousrecombination between genomes from these subspecies.

[0155] The PCR reactions (25 μL) were carried out over 35 cycles (15seconds at 94° C., 30 seconds at 55° C. and 30 seconds at 72° C.), with0.25 U Taq DNA polymerase and 0.6 μg genomic DNA in reaction buffercontaining 2 mM MgCl₂. PCR products were separated by agarose gelelectrophoresis (4% ultra high resolution agarose) and visualised byethidiumbromide staining. The results from the PCR experiments aresummarised in Table 1, which also shows the sequence of PCR primers,primer annealing temperature (Tm), PCR product length and chromosomelocation of SSLP (with respect to the R1 map of May 1997, Weeds World4i).

[0156] F. Production of Hybrid Plants

[0157] C24 plants heterozygous for chlorina3A:AclGUS are crossed as maleto emasculated wildtype Ler to produce Ler/C24(chlorina3A, GUS) hybridseeds.

[0158] Due to the heterozygosity of the C24 parent, only 50% of hybridplants have inherited the chlorina3A:Ac/GUS locus. The remaining 50% ofhybrid plants are wildtype with respect to chlorina3A:Ac/GUS. Since themutant locus is linked to a kanamycin resistance gene (contained on thesame T-DNA as GUS) mutant plants can be pre-selected by germinatinghybrid seeds on germination medium containing 50 mg/L kanamycin.

[0159] Ler plants homozygous for the five chromosome markers are malesterile (ms1-1) and are crossed without emasculation to wildtype C24 toproduce Ler (an-1, py-1, gl1-1, cer2-1, ms1-1) C24 hybrid seeds.

[0160] Other Ler plants, which are male fertile, are crossed afteremasculation of the female parent to produce Ler/C24 hybrid seeds.

[0161] G. Introduction of MSH3 and MSH6/3 Antisense Genes intoArabidopsis and Analysis of Meiotic Homeologous Recombination

[0162] (i) Transformation of Hybrid Plants and Analysis of HomeologousMeiotic Recombination

[0163] The plant transformation vectors comprising the antisense genesdescribed in (B) and (C) above are introduced into Agrobacteriumtumefaciens strain AGL1 (Lazo et al. 1991, Bio/Technology 9, 963-967) byelectroporation. Recombinant Agrobacterium clones are selected on LBmedium containing 50 mg/L rifampicin and 100 mg/L carbenicillin.Selected clones are used to infect roots of Arabidopsis hybrid plants(described in (F) above) using the root transformation protocol ofValvekens et al. (1988, PNAS 85. 5536-5540) except that shoot and rootinducing media contain hygromycin (10 mg/L) instead of kanamycin.

[0164] Plants regenerated from roots of hybrid plants are genetic clonesof root donating plants and therefore are again genetic hybrids of twoArabidopsis subspecies described in (F). However, in contrast to theroot donating plants, the regenerated hybrid plants also contain theintroduced transgene and the co-introduced hygromycin resistance genewith the latter allowing these plants to grow on hygromycin containingculture medium.

[0165] Hygromycin resistant plants are then allowed to enter thereproductive phase and to produce gametes by meiotic divisions ofmicrospore and megaspore mothercells. At meiosis, the DMC1 promoter isactivated and can direct the expression of antisense genes described in(B) and (C) above, leading to decreased MSH6 and/or MSH3 geneexpression. This in turn depletes the gamete mothercells of MSH6 and/orMSH3 protein, thus causing alteration of MMR during meiotic divisionsand increasing the recombination frequency between homeologouschromosomes.

[0166] Transgenic plants are then allowed to self-fertilise and toproduce seeds. These seeds (F2 seeds with respect to hybrid production),and the plants derived therefrom, carry the homeologous recombinationevents which can be identified by using the visual and molecularrecombination markers described in (E) above.

[0167] In case of homeologous recombination between chromosomes of Lerand C24(chlorina3A:Ac, GUS), the analysis concentrates on chromosome 2by selecting plants showing the visual phenotypic marker chlorina. Thismarker thus serves as a reference point as it indicates that respectivechromosomes 2 originate from C24. Other markers, such as GUS ormolecular markers, on chromosome 2 may then be used to identifychromosomal regions which are derived from the Ler chromosome as aresult of homeologous recombination. F2 plants of control transformantsnot expressing the antisense gene(s) can be analysed in parallel and theresults can be used for comparison to homeologous recombination resultsobtained in antisense plants.

[0168] (ii) Transformation of C24 Wildtype, Hybrid Plant Production andAnalysis of Homeologous Meiotic Recombination

[0169] Introduction of MMR altering genes into wildtype C24 is doneusing the root transformation protocol as described in G (i) fortransformation of hybrid plants. Transformed plants are selected byresistance to either 10 mg/L hygromycin (in case of transformation withT-DNA's derived from pNOS-Hyg-SCV) or to 50 mg/L kanamycin (in case oftransformation with T-DNA's derived from pBIN19).

[0170] Transgenic plants are then allowed to self-fertilise and toproduce seeds (T1 seeds). Segregation of the antibiotic resistance genein the T1 population then indicates the number of transgene loci. Lineswith a single transgene locus (indicated by a 3:1 ratio ofresistant:sensitive plants) are selected and are bred to homozygosity.This is done by collecting selfed seeds (T2) from T1 plants andsubsequent testing of at least four independent T2 seed populations forsegregation of the antibiotic resistance gene. T2 populations which donot segregate the antibiotic resistance gene are assumed to behomozygous for both the resistance gene and the linked MMR alteringgene.

[0171] C24 plants homozygous for the MMR altering gene are then crossedto Ler lines homozygous for recessive visual markers (see E (ii)) toproduce C24/Ler hybrid plants as described in (F). These F1 hybrids arethen allowed to enter the reproductive phase and to produce gametes bymeiotic division of microspore and megaspore mothercells. At meiosis,the DMC 1 promoter is activated and can direct the expression ofantisense or sense genes described in (B), (C) and (D) above, leading todecreased MSH6 and/or MSH3 gene expression. This in turn depletes thegamete mothercells of MSH6 and/or MSH3 protein, thus causing alterationof MMR during meiotic divisions and increasing the recombinationfrequency between the homeologous chromosomes of C24 and Ler.Recombination events are then scored in the F2 generation.

[0172] For recombination analysis, the hybrid plants are allowed toself-fertilise and to produce F2 seeds. F2 plants are then preselectedfor a first visual marker. Since this marker is recessive, its visualpresence indicates homozygosity for Ler DNA at the relevant locus. ThoseF2 plants which show this first visual marker are then analysed for thepresence or absence of a second visual marker which in the Ler parent isclosely linked to the first marker. Absence of the second visual markerindicates recombination between the relevant C24 and Ler chromosomesbetween the first and second marker. The frequency of recombination intransgenic hybrids is compared to the recombination frequency in controlhybrids not expressing the MMR altering gene.

[0173] Examples of recombination analysis are the following.

[0174] The Ler line NW22(dis1, ga4, th1) is used for crosses totransformedC24.

[0175] F2 plants are preselected first for thiamine requirement (th1)and then are further analysed for re-appearance of wildtype height (lossof ga4) and/or re-appearance of normal trichomes (loss of dis1) as aresult of recombination.

[0176] The Ler line NW10(tz-201, cer3) is used for crosses totransformedC24.

[0177] F2 plants are then preselected first for thiazole requirement(tz) and then are further analysed for re-appearance of normal. i.e.non-shiny stems (loss of cer3) as a result of recombination.

[0178] The Ler line NW134 (ttg, ga3) is used for crosses totransformedC24. F2 plants are first preselected for dwarfish appearance(ga3) and are then analysed for re-appearance of trichomes (loss of ttg)as a result of recombination.

[0179] Ler plants homozygous for abi3-1 and gl1-1 are used for crossesto transformedC24. F2 plants are first preselected for their ability togerminate in the presence of abscisic acid and are then analysed forre-appearance of trichomes on the leaves (loss of gll-1) as a result ofrecombination.

[0180] In the case of homeologous recombination between transformedC24and the Ler line NW1 (an-1, py-1, gl1-1, cer2-1, ms1-1), recombinationanalysis is similar the one described above, except that the secondmarker is not a visual marker but has to be a molecular marker. This isbecause the Ler parent carries only one visual marker per chromosome.TABLE 1 SSLP Markers in Arabidopsis thaliana Subspecies RI Map PCRPrimer Chromosome Position Pair Primer Sequence Tm [° C.] length/COLlength/LER length/C24 I 2.3 ATEAT1 F GCCACTGCGTGAATGATATG 57.8 172 162162 ATEAT1 R CGAACAGCCAACATTAATTCCC 58.2 I 9.3 NGA63 FAACCAAGGCACAGAAGCG 57.3 111 89 120 NGA63 R ACCCAAGTGATCGCCACC 59.6 I40.1 NGA248 F TACCGAACCAAAACACAAAGG 56.1 143 129 no amplific. NGA248 RTCTGTATCTCGGTGAATTCTCC 58.2 I 81.2 NGA128 F GGTCTGTTGATGTCGTAAGTCG 60.1180 190 no amplific. NGA128 R ATCTTGAAACCTTTAGGGAGGG 58.2 I 81.2 NGA280F CTGATCTCACGGACAATAGTGC 60.1 105 85 85 NGA280 R GGCTCCATAAAAAGTGCACC57.8 I 111.4 NGA111 F CTCCAGTTGGAAGCTAAAGGG 60 128 162 170 NGA111 RTGTTTTTTAGGACAAATGGCG 70 II ca. 7.5 NGA168 F CCTTCACATCCAAAACCCAC 57.8213 217 208 NGA168 R GCACATACCCACAACCAGAA 57.8 II ca. 48 NGA1126LCGCTACGCTTTTCGGTAAAG 57.8 191 199 196 NGA1126R GCACAGTCCAAGTCACAACC 59.9II 62.2 NGA361L AAAGAGATGAGAATTTGGAC 51.7 114 120 114 NGA361RACATATCAATATATTAAAGTAGC 49.5 II 73 NGA168 F TCGTCTACTGCACTGCCG 59.6 151135 135 NGA168 R GAGGACATGTATAGGAGCCTCG 61.9 II ca. 77 AthBIO2 LTGACCTCCTCTTCCATGGAG 59.9 141 209 139 AthBIO2 R TTAACAGAAACCCAAAGCTTTC54.5 II ca. 83 AthUBIQUE L AGGCAAATGTCCATTTCATTG 54.1 146 148 148AthUBIQUE R ACGACATGGCAGATTTCTCC 57.8 III 3.4 NGA172 FAGCTGCTTCCTTATAGCGTCC 60 162 136 140 NGA172 R CATCCGAATGCCATTGTTC 55.4III 12.8 NGA126 F GAAAAAACGCTACTTTCGTGG 56.1 119 147 no amplific. NGA126R CAAGAGCAATATCAAGAGCAGC 58.2 III 17.5 NGA162 F CATGCAATTTGCATCTGAGG55.8 107 89 no amplilic. NGA162 R CTCTGTCACTCTTTTCCTCTGG 60.1 III 81.8NGA6 F TGGATTTCTTCCTCTCTTCAC 56.1 143 123 143 NGA6 RATGGAGAAGCTTACACTGATC 56.1 IV 19.8 NGA12 F AATGTTGTCCTCCCCTCCTC 59.9 247234 220 NGA12 R TGATGCTCTCTGAAACAAGAGC 58.2 IV 24.1 NGA8 FGAGGGCAAATCTTTATTTCGG 56.1 154 198 190 NGA8 R TGGCTTTCGTTTATAAACATCC54.5 IV 102 NGA1107 L GCGAAAAAACAAAAAAATCCA 50.2 150 140 140 NGA1107 RCGACGAATCGACAGAATTAGG 58 V 11.8 NGA225 F GAAATCCAAATCCCAGAGAGG 58 119189 119 NGA225 R TCTCCCCACTAGTTTTGTGTCC 60.1 V 16.7 NGA249 FTACCGTCAATTTCATCGCC 55.4 125 115 115 NGA249 R GGATCCCTAACTGTAAAATCCC58.2 V 19.9 CA72 F AATCCCAGTAACCAAACACACA 56.3 124 110 110 CA72 RCCCAGTCTAACCACGACCAC 61.9 V 20 NGA151 F GTTTTGGGAAGTTTTGCTGG 55.8 150120 130 NGA151 R CAGTCTAAAAGCGAGAGTATGATG 58.6 V 24 NGA106 FGTTATGGAGTTTCTAGGGCACG 60.1 157 123 130 NGA106 R TGCCCCATTTTGTTCTTCTC55.8 V 37.8 NGA139 F AGAGCTACCAGATCCGATGG 59.9 174 132 132 NGA139 RGGTTTCGTTTCACTATCCAGG 55.8 V 50 NGA76 F GGAGAAAATGTCACTCTCCACC 60.1231 >250 300 NGA76 R AGGCATGGGAGACATTTACG 57.8 V 61.1 ATHSO191 LCTCCACCAATCATGCAAATG 55.8 148 156 146 ATHSO191 R TGATGTTGATGGAGATGGTCA53.7 V 81.7 NGA129 F TCAGGAGGAACTAAAGTGAGGG 60.1 177 179 172 NGA129 RCACACTGAAGATGGTCTTGAGG 60.1

[0181]

1 103 1 23 DNA Artificial sequence modified_base 11 I 1 ctggatccacnggnccnaay atg 23 2 23 DNA Artificial sequence modified_base 15 I 2ctggatccrt artgngtnrc raa 23 3 24 DNA Artificial sequence MSH3 specificprimer 636 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 3tgctagtgcc tcttgcaagc tcat 24 4 27 DNA Artificial sequence Primer AP1for PCR using cDNA of Arabidopsis thaliana ecotype Columbia containingadapter sequences ligated to both its ends 4 ccatcctaat acgactcactatagggc 27 5 23 DNA Artificial sequence Primer AP2 for PCR using cDNA ofArabidopsis thaliana ecotype Columbia containing adapter sequencesligated to both its ends 5 actcactata gggctcgagc ggc 23 6 30 DNAArtificial sequence MSH3 specific primer S525 for PCR using cDNA ofArabidopsis thaliana ecotype Columbia 6 aggttctgat tatgtgtgac gctttactta30 7 29 DNA Artificial sequence MSH3 specific primer S51 for PCR usingcDNA of Arabidopsis thaliana ecotype Columbia 7 ggatcgggta ctgggttttgagtgtgagg 29 8 24 DNA Artificial sequence MSH3 specific primer 635 forPCR using cDNA of Arabidopsis thaliana ecotype Columbia 8 gcacgtgcttgatggtgttt tcac 24 9 28 DNA Artificial sequence MSH3 specific primerS523 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 9tcagacagta tccagcatgg cagaagta 28 10 33 DNA Artificial sequence MSH3specific primer 1S5 for PCR using cDNA of Arabidopsis thaliana ecotypeColumbia 10 atcccgggat gggcaagcaa aagcagcaga cga 33 11 27 DNA Artificialsequence MSH3 specific primer S53 for PCR using cDNA of Arabidopsisthaliana ecotype Columbia 11 gacaaagagc gaaatgaggc cccttgg 27 12 1250DNA Arabidopsis thaliana ecotype Columbia Clone 52 12 cccgggatgggcaagcaaaa gcagcagacg atttctcgtt tcttcgctcc caaacccaaa 60 tccccgactcacgaaccgaa tccggtagcc gaatcatcaa caccgccacc gaagatatcc 120 gccactgtatccttctctcc ttccaagcgt aagcttctct ccgaccacct cgccgccgcg 180 tcacccaaaaagcctaaact ttctcctcac actcaaaacc cagtacccga tcccaattta 240 caccaaagatttctccagag atttctggaa ccctcgccgg aggaatatgt tcccgaaacg 300 tcatcatcgaggaaatacac accattggaa cagcaagtgg tggagctaaa gagcaagtac 360 ccagatgtggttttgatggt ggaagttggt tacaggtaca gattcttcgg agaagacgcg 420 gagatcgcagcacgcgtgtt gggtatttac gctcatatgg atcacaattt catgacggcg 480 agtgtgccaacatttcgatt gaatttccat gtgagaagac tggtgaatgc aggatacaag 540 attggtgtagtgaagcagac tgaaactgca gccattaagt cccatggtgc aaaccggacc 600 ggcccttttttccggggact gtcggcgttg tataccaaag ccacgcttga agcggctgag 660 gatataagtggtggttgtgg tggtgaagaa ggttttggtt cacagagtaa tttcttggtt 720 tgtgttgtggatgagagagt taagtcggag acattaggct gtggtattga aatgagtttt 780 gatgttagagtcggtgttgt tggcgttgaa atttcgacag gtgaagttgt ttatgaagag 840 ttcaatgataatttcatgag aagtggatta gaggctgtga ttttgagctt gtcaccagct 900 gagctgttgcttggccagcc tctttcacaa caaactgaga agtttttggt ggcacatgct 960 ggacctacctcaaacgttcg agtggaacgt gcctcactgg attgtttcag caatggtaat 1020 gcagtagatgaggttatttc attatgtgaa aaaatcagcg caggtaactt agaagatgat 1080 aaagaaatgaagctggaggc tgctgaaaaa ggaatgtctt gcttgacagt tcatacaatt 1140 atgaacatgccacatctgac tgttcaagcc ctcgccctaa cgttttgcca tctcaaacag 1200 tttggatttgaaaggatcct ttaccaaggg gcctcatttc gctctttg 1250 13 34 DNA Artificialsequence MSH3 specific primer 2S5 for PCR using cDNA of Arabidopsisthaliana ecotype Columbia 13 atcccgggtc aaaatgaaca agttggtttt agtc 34 1427 DNA Artificial sequence MSH3 specific primer S52 for PCR using cDNAof Arabidopsis thaliana ecotype Columbia 14 gccacatctg actgttcaagccctcgc 27 15 2110 DNA Arabidopsis thaliana ecotype Columbia Clone 13 15gccacatctg actgttcaag ccctcgccct aacgttttgc catctcaaac agtttggatt 60tgaaaggatc ctttaccaag gggcctcatt tcgctctttg tcaagtaaca cagagatgac 120tctctcagcc aatactctgc aacagttgga ggttgtgaaa aataattcag atggatcgga 180atctggctcc ttattccata atatgaatca cacacttaca gtatatggtt ccaggcttct 240tagacactgg gtgactcatc ctctatgcga tagaaatttg atatctgctc ggcttgatgc 300tgtttctgag atttctgctt gcatgggatc tcatagttct tcccagctca gcagtgagtt 360ggttgaagaa ggttctgaga gagcaattgt atcacctgag ttttatctcg tgctctcctc 420agtcttgaca gctatgtcta gatcatctga tattcaacgt ggaataacaa gaatctttca 480tcggactgct aaagccacag agttcattgc agttatggaa gctattttac ttgcggggaa 540gcaaattcag cggcttggca taaagcaaga ctctgaaatg aggagtatgc aatctgcaac 600tgtgcgatct actcttttga gaaaattgat ttctgttatt tcatcccctg ttgtggttga 660caatgccgga aaacttctct ctgccctaaa taaggaagcg gctgttcgag gtgacttgct 720cgacatacta atcacttcca gcgaccaatt tcctgagctt gctgaagctc gccaagcagt 780tttagtcatc agggaaaagc tggattcctc gatagcttca tttcgcaaga agctcgctat 840tcgaaatttg gaatttcttc aagtgtcggg gatcacacat ttgatagagc tgcccgttga 900ttccaaggtc cctatgaatt gggtgaaagt aaatagcacc aagaagacta ttcgatatca 960tcccccagaa atagtagctg gcttggatga gctagctcta gcaactgaac atcttgccat 1020tgtgaaccga gcttcgtggg atagtttcct caagagtttc agtagatact acacagattt 1080taaggctgcc gttcaagctc ttgctgcact ggactgtttg cactcccttt caactctatc 1140tagaaacaag aactatgtcc gtcccgagtt tgtggatgac tgtgaaccag ttgagataaa 1200catacagtct ggtcgtcatc ctgtactgga gactatatta caagataact tcgtcccaaa 1260tgacacaatt ttgcatgcag aaggggaata ttgccaaatt atcaccggac ctaacatggg 1320aggaaagagc tgctatatcc gtcaagttgc tttaatttcc ataatggctc aggttggttc 1380ctttgtacca gcgtcattcg ccaagctgca cgtgcttgat ggtgttttca ctcggatggg 1440tgcttcagac agtatccagc atggcagaag tacctttcta gaagaattaa gtgaagcgtc 1500acacataatc agaacctgtt cttctcgttc gcttgttata ttagatgagc ttggaagagg 1560cactagcaca cacgacggtg tagccattgc ctatgcaaca ttacagcatc tcctagcaga 1620aaagagatgt ttggttcttt ttgtcacgca ttaccctgaa atagctgaga tcagtaacgg 1680attcccaggt tctgttggga cataccatgt ctcgtatctg acattgcaga aggataaagg 1740cagttatgat catgatgatg tgacctacct atataagctt gtgcgtggtc tttgcagcag 1800gagctttggt tttaaggttg ctcagcttgc ccagatacct ccatcatgta tacgtcgagc 1860catttcaatg gctgcaaaat tggaagctga ggtacgtgca agagagagaa atacacgcat 1920gggagaacca gaaggacatg aagaaccgag aggcgcagaa gaatctattt cggctctagg 1980tgacttgttt gcagacctga aatttgctct ctctgaagag gacccttgga aagcattcga 2040gtttttaaag catgcttgga agattgctgg caaaatcaga ctaaaaccaa cttgttcatt 2100ttgacccggg 2110 16 29 DNA Artificial sequence MSH3 specific primer S51for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 16ggatcgggta ctgggttttg agtgtgagg 29 17 30 DNA Artificial sequence MSH3specific primer S525 for PCR using cDNA of Arabidopsis thaliana ecotypeColumbia 17 aggttctgat tatgtgtgac gctttactta 30 18 3522 DNA Arabidopsisthaliana ecotype Columbia CDS (100)....(3342) AtMSH3 full-length cDNAand deduced sequence of the encoded polypeptide 18 cctaagaaag cgcgcgaaaattggcaaccc aagttcgcca tagccacgac cacgaccttc 60 catttctctt aaacggaggagattacgaat aaagcaatt 99 atg ggc aag caa aag cag cag acg att tct cgt ttcttc gct ccc aaa 147 Met Gly Lys Gln Lys Gln Gln Thr Ile Ser Arg Phe PheAla Pro Lys 1 5 10 15 ccc aaa tcc ccg act cac gaa ccg aat ccg gta gccgaa tca tca aca 195 Pro Lys Ser Pro Thr His Glu Pro Asn Pro Val Ala GluSer Ser Thr 20 25 30 ccg cca ccg aag ata tcc gcc act gta tcc ttc tct ccttcc aag cgt 243 Pro Pro Pro Lys Ile Ser Ala Thr Val Ser Phe Ser Pro SerLys Arg 35 40 45 aag ctt ctc tcc gac cac ctc gcc gcc gcg tca ccc aaa aagcct aaa 291 Lys Leu Leu Ser Asp His Leu Ala Ala Ala Ser Pro Lys Lys ProLys 50 55 60 ctt tct cct cac act caa aac cca gta ccc gat ccc aat tta caccaa 339 Leu Ser Pro His Thr Gln Asn Pro Val Pro Asp Pro Asn Leu His Gln65 70 75 80 aga ttt ctc cag aga ttt ctg gaa ccc tcg ccg gag gaa tat gttccc 387 Arg Phe Leu Gln Arg Phe Leu Glu Pro Ser Pro Glu Glu Tyr Val Pro85 90 95 gaa acg tca tca tcg agg aaa tac aca cca ttg gaa cag caa gtg gtg435 Glu Thr Ser Ser Ser Arg Lys Tyr Thr Pro Leu Glu Gln Gln Val Val 100105 110 gag cta aag agc aag tac cca gat gtg gtt ttg atg gtg gaa gtt ggt483 Glu Leu Lys Ser Lys Tyr Pro Asp Val Val Leu Met Val Glu Val Gly 115120 125 tac agg tac aga ttc ttc gga gaa gac gcg gag atc gca gca cgc gtg531 Tyr Arg Tyr Arg Phe Phe Gly Glu Asp Ala Glu Ile Ala Ala Arg Val 130135 140 ttg ggt att tac gct cat atg gat cac aat ttc atg acg gcg agt gtg579 Leu Gly Ile Tyr Ala His Met Asp His Asn Phe Met Thr Ala Ser Val 145150 155 160 cca aca ttt cga ttg aat ttc cat gtg aga aga ctg gtg aat gcagga 627 Pro Thr Phe Arg Leu Asn Phe His Val Arg Arg Leu Val Asn Ala Gly165 170 175 tac aag att ggt gta gtg aag cag act gaa act gca gcc att aagtcc 675 Tyr Lys Ile Gly Val Val Lys Gln Thr Glu Thr Ala Ala Ile Lys Ser180 185 190 cat ggt gca aac cgg acc ggc cct ttt ttc cgg gga ctg tcg gcgttg 723 His Gly Ala Asn Arg Thr Gly Pro Phe Phe Arg Gly Leu Ser Ala Leu195 200 205 tat acc aaa gcc acg ctt gaa gcg gct gag gat ata agt ggt ggttgt 771 Tyr Thr Lys Ala Thr Leu Glu Ala Ala Glu Asp Ile Ser Gly Gly Cys210 215 220 ggt ggt gaa gaa ggt ttt ggt tca cag agt aat ttc ttg gtt tgtgtt 819 Gly Gly Glu Glu Gly Phe Gly Ser Gln Ser Asn Phe Leu Val Cys Val225 230 235 240 gtg gat gag aga gtt aag tcg gag aca tta ggc tgt ggt attgaa atg 867 Val Asp Glu Arg Val Lys Ser Glu Thr Leu Gly Cys Gly Ile GluMet 245 250 255 agt ttt gat gtt aga gtc ggt gtt gtt ggc gtt gaa att tcgaca ggt 915 Ser Phe Asp Val Arg Val Gly Val Val Gly Val Glu Ile Ser ThrGly 260 265 270 gaa gtt gtt tat gaa gag ttc aat gat aat ttc atg aga agtgga tta 963 Glu Val Val Tyr Glu Glu Phe Asn Asp Asn Phe Met Arg Ser GlyLeu 275 280 285 gag gct gtg att ttg agc ttg tca cca gct gag ctg ttg cttggc cag 1011 Glu Ala Val Ile Leu Ser Leu Ser Pro Ala Glu Leu Leu Leu GlyGln 290 295 300 cct ctt tca caa caa act gag aag ttt ttg gtg gca cat gctgga cct 1059 Pro Leu Ser Gln Gln Thr Glu Lys Phe Leu Val Ala Met Ala GlyPro 305 310 315 320 acc tca aac gtt cga gtg gaa cgt gcc tca ctg gat tgtttc agc aat 1107 Thr Ser Asn Val Arg Val Glu Arg Ala Ser Leu Asp Cys PheSer Asn 325 330 335 ggt aat gca gta gat gag gtt att tca tta tgt gaa aaaatc agc gca 1155 Gly Asn Ala Val Asp Glu Val Ile Ser Leu Cys Glu Lys IleSer Ala 340 345 350 ggt aac tta gaa gat gat aaa gaa atg aag ctg gag gctgct gaa aaa 1203 Gly Asn Leu Glu Asp Asp Lys Glu Met Lys Leu Glu Ala AlaGlu Lys 355 360 365 gga atg tct tgc ttg aca gtt cat aca att atg aac atgcca cat ctg 1251 Gly Met Ser Cys Leu Thr Val His Thr Ile Met Asn Met ProHis Leu 370 375 380 act gtt caa gcc ctc gcc cta acg ttt tgc cat ctc aaacag ttt gga 1299 Thr Val Gln Ala Leu Ala Leu Thr Phe Cys His Leu Lys GlnPhe Gly 385 390 395 400 ttt gaa agg atc ctt tac caa ggg gcc tca ttt cgctct ttg tca agt 1347 Phe Glu Arg Ile Leu Tyr Gln Gly Ala Ser Phe Arg SerLeu Ser Ser 405 410 415 aac aca gag atg act ctc tca gcc aat act ctg caacag ttg gag gtt 1395 Asn Thr Glu Met Thr Leu Ser Ala Asn Thr Leu Gln GlnLeu Glu Val 420 425 430 gtg aaa aat aat tca gat gga tcg gaa tct ggc tcctta ttc cat aat 1443 Val Lys Asn Asn Ser Asp Gly Ser Glu Ser Gly Ser LeuPhe His Asn 435 440 445 atg aat cac aca ctt aca gta tat gct tcc agg cttctt aga cac tgg 1491 Met Asn His Thr Leu Thr Val Tyr Gly Ser Arg Leu LeuArg His Trp 450 455 460 gtg act cat cct cta tgc gat aga aat ttg ata tctgct cgg ctt gat 1539 Val Thr His Pro Leu Cys Asp Arg Asn Leu Ile Ser AlaArg Leu Asp 465 470 475 480 gct gtt tct gag att tct gct tgc atg gga tctcat agt tct tcc cag 1587 Ala Val Ser Glu Ile Ser Ala Cys Met Gly Ser HisSer Ser Ser Gln 485 490 495 ctc agc agt gag ttg gtt gaa gaa ggt tct gagaga gca att gta tca 1635 Leu Ser Ser Glu Leu Val Glu Glu Gly Ser Glu ArgAla Ile Val Ser 500 505 510 cct gag ttt tat ctc gtg ctc tcc tca gtc ttgaca gct atg tct aga 1683 Pro Glu Phe Tyr Leu Val Leu Ser Ser Val Leu ThrAla Met Ser Arg 515 520 525 tca tct gat att caa cgt gga ata aca aga atcttt cat cgg act gct 1731 Ser Ser Asp Ile Gln Arg Gly Ile Thr Arg Ile PheHis Arg Thr Ala 530 535 540 aaa gcc aca gag ttc att gca gtt atg gaa gctatt tta ctt gcg ggg 1779 Lys Ala Thr Glu Phe Ile Ala Val Met Glu Ala IleLeu Leu Ala Gly 545 550 555 560 aag caa att cag cgg ctt ggc ata aag caagac tct gaa atg agg agt 1827 Lys Gln Ile Gln Arg Leu Gly Ile Lys Gln AspSer Glu Met Arg Ser 565 570 575 atg caa tct gca act gtg cga tct act cttttg aga aaa ttg att tct 1875 Met Gln Ser Ala Thr Val Arg Ser Thr Leu LeuArg Lys Leu Ile Ser 580 585 590 gtt att tca tcc cct gtt gtg gtt gac aatgcc gga aaa ctt ctc tct 1923 Val Ile Ser Ser Pro Val Val Val Asp Asn AlaGly Lys Leu Leu Ser 595 600 605 gcc cta aat aag gaa gcg gct gtt cga ggtgac ttg ctc gac ata cta 1971 Ala Leu Asn Lys Glu Ala Ala Val Arg Gly AspLeu Leu Asp Ile Leu 610 615 620 atc act tcc agc gac caa ttt cct gag cttgct gaa gct cgc caa gca 2019 Ile Thr Ser Ser Asp Gln Phe Pro Glu Leu AlaGlu Ala Arg Gln Ala 625 630 635 640 gtt tta gtc atc agg gaa aag ctg gattcc tcg ata gct tca ttt cgc 2067 Val Leu Val Ile Arg Glu Lys Leu Asp SerSer Ile Ala Ser Phe Arg 645 650 655 aag aag ctc gct att cga aat ttg gaattt ctt caa gtg tcg ggg atc 2115 Lys Lys Leu Ala Ile Arg Asn Leu Glu PheLeu Gln Val Ser Gly Ile 660 665 670 aca cat ttg ata gag ctg ccc gtt gattcc aag gtc cct atg aat tgg 2163 Thr His Leu Ile Glu Leu Pro Val Asp SerLys Val Pro His Asn Trp 675 680 685 gtg aaa gta aat agc acc aag aag actatt cga tat cat ccc cca gaa 2211 Val Lys Val Asn Ser Thr Lys Lys Thr IleArg Tyr His Pro Pro Glu 690 695 700 ata gta gct ggc ttg gat gag cta gctcta gca act gaa cat ctt gcc 2259 Ile Val Ala Gly Leu Asp Glu Leu Ala LeuAla Thr Glu His Leu Ala 705 710 715 720 att gtg aac cga gct tcg tgg gatagt ttc ctc aag agt ttc agt aga 2307 Ile Val Asn Arg Ala Ser Trp Asp SerPhe Leu Lys Ser Phe Ser Arg 725 730 735 tac tac aca gat ttt aag gct gccgtt caa gct ctt gct gca ctg gac 2355 Tyr Tyr Thr Asp Phe Lys Ala Ala ValGln Ala Leu Ala Ala Leu Asp 740 745 750 tgt ttg cac tcc ctt tca act ctatct aga aac aag aac tat gtc cgt 2403 Cys Leu His Ser Leu Ser Thr Leu SerArg Asn Lys Asn Tyr Val Arg 755 760 765 ccc gag ttt gtg gat gac tgt gaacca gtt gag ata aac ata cag tct 2451 Pro Glu Phe Val Asp Asp Cys Glu ProVal Glu Ile Asn Ile Gln Ser 770 775 780 ggt cgt cat cct gta ctg gag actata tta caa gat aac ttc gtc cca 2499 Gly Arg His Pro Val Leu Glu Thr IleLeu Gln Asp Asn Phe Val Pro 785 790 795 800 aat gac aca att ttg cat gcagaa ggg gaa tat tgc caa att atc acc 2547 Asn Asp Thr Ile Leu His Ala GluGly Glu Tyr Cys Gln Ile Ile Thr 805 810 815 gga cct aac atg gga gga aagagc tgc tat atc cgt caa gtt gct tta 2595 Gly Pro Asn Met Gly Gly Lys SerCys Tyr Ile Arg Gln Val Ala Leu 820 825 830 att tcc ata atg gct cag gttggt tcc ttt gta cca gcg tca ttc gcc 2643 Ile Ser Ile Met Ala Gln Val GlySer Phe Val Pro Ala Ser Phe Ala 835 840 845 aag ctg cac gtg ctt gat ggtgtt ttc act cgg atg ggt gct tca gac 2691 Lys Leu His Val Leu Asp Gly ValPhe Thr Arg Met Gly Ala Ser Asp 850 855 860 agt atc cag cat ggc aga agtacc ttt cta gaa gaa tta agt gaa gcg 2739 Ser Ile Gln His Gly Arg Ser ThrPhe Leu Glu Glu Leu Ser Glu Ala 865 870 875 880 tca cac ata atc aga acctgt tct tct cgt tcg ctt gtt ata tta gat 2787 Ser His Ile Ile Arg Thr CysSer Ser Arg Ser Leu Val Ile Leu Asp 885 890 895 gag ctt gga aga ggc actagc aca cac gac ggt gta gcc att gcc tat 2835 Glu Leu Gly Arg Gly Thr SerThr His Asp Gly Val Ala Ile Ala Tyr 900 905 910 gca aca tta cag cat ctccta gca gaa aag aga tgt ttg gtt ctt ttt 2883 Ala Thr Leu Gln His Leu LeuAla Glu Lys Arg Cys Leu Val Leu Phe 915 920 925 gtc acg cat tac cct gaaata gct gag atc agt aac gga ttc cca ggt 2931 Val Thr His Tyr Pro Glu IleAla Glu Ile Ser Asn Gly Phe Pro Gly 930 935 940 tct gtt ggg aca tac catgtc tcg tat ctg aca ttg cag aag gat aaa 2979 Ser Val Gly Thr Tyr His ValSer Tyr Leu Thr Leu Gln Lys Asp Lys 945 950 955 960 ggc agt tat gat catgat gat gtg acc tac cta tat aag ctt gtg cgt 3027 Gly Ser Tyr Asp His AspAsp Val Thr Tyr Leu Tyr Lys Leu Val Arg 965 970 975 ggt ctt tgc agc aggagc ttt ggt ttt aag gtt gct cag ctt gcc cag 3075 Gly Leu Cys Ser Arg SerPhe Gly Phe Lys Val Ala Gln Leu Ala Gln 980 985 990 ata cct cca tca tgtata cgt cga gcc att tca atg gct gca aaa ttg 3123 Ile Pro Pro Ser Cys IleArg Arg Ala Ile Ser Met Ala Ala Lys Leu 995 1000 1005 gaa gct gag gtacgt gca aga gag aga aat aca cgc atg gga gaa cca 3171 Glu Ala Glu Val ArgAla Arg Glu Arg Asn Thr Arg Met Gly Glu Pro 1010 1015 1020 gaa gga catgaa gaa ccg aga ggc gca gaa gaa tct att tcg gct cta 3219 Glu Gly His GluGlu Pro Arg Gly Ala Glu Glu Ser Ile Ser Ala Leu 1025 1030 1035 1040 ggtgac ttg ttt gca gac ctg aaa ttt gct ctc tct gaa gag gac cct 3267 Gly AspLeu Phe Ala Asp Leu Lys Phe Ala Leu Ser Glu Glu Asp Pro 1045 1050 1055tgg aaa gca ttc gag ttt tta aag cat gct tgg aag att gct ggc aaa 3315 TrpLys Ala Phe Glu Phe Leu Lys His Ala Trp Lys Ile Ala Gly Lys 1060 10651070 atc aga cta aaa cca act tgt tca ttt tgatttaatc ttaacattat 3362 IleArg Leu Lys Pro Thr Cys Ser Phe 1075 1080 agcaactgca aggtcttgatcatctgttag ttgcgtacta acttatgtgt attagtataa 3422 caagaaaaga gaattagagagatggattct aatccggtgt tgcagtacat cttttctcca 3482 cccgcataaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 3522 19 1081 PRT Arabidopsis thaliana ecotypeColumbia Polypeptide MSH3 19 Met Gly Lys Gln Lys Gln Gln Thr Ile Ser ArgPhe Phe Ala Pro Lys 1 5 10 15 Pro Lys Ser Pro Thr His Glu Pro Asn ProVal Ala Glu Ser Ser Thr 20 25 30 Pro Pro Pro Lys Ile Ser Ala Thr Val SerPhe Ser Pro Ser Lys Arg 35 40 45 Lys Leu Leu Ser Asp His Leu Ala Ala AlaSer Pro Lys Lys Pro Lys 50 55 60 Leu Ser Pro His Thr Gln Asn Pro Val ProAsp Pro Asn Leu His Gln 65 70 75 80 Arg Phe Leu Gln Arg Phe Leu Glu ProSer Pro Glu Glu Tyr Val Pro 85 90 95 Glu Thr Ser Ser Ser Arg Lys Tyr ThrPro Leu Glu Gln Gln Val Val 100 105 110 Glu Leu Lys Ser Lys Tyr Pro AspVal Val Leu Met Val Glu Val Gly 115 120 125 Tyr Arg Tyr Arg Phe Phe GlyGlu Asp Ala Glu Ile Ala Ala Arg Val 130 135 140 Leu Gly Ile Tyr Ala HisMet Asp His Asn Phe Met Thr Ala Ser Val 145 150 155 160 Pro Thr Phe ArgLeu Asn Phe His Val Arg Arg Leu Val Asn Ala Gly 165 170 175 Tyr Lys IleGly Val Val Lys Gln Thr Glu Thr Ala Ala Ile Lys Ser 180 185 190 His GlyAla Asn Arg Thr Gly Pro Phe Phe Arg Gly Leu Ser Ala Leu 195 200 205 TyrThr Lys Ala Thr Leu Glu Ala Ala Glu Asp Ile Ser Gly Gly Cys 210 215 220Gly Gly Glu Glu Gly Phe Gly Ser Gln Ser Asn Phe Leu Val Cys Val 225 230235 240 Val Asp Glu Arg Val Lys Ser Glu Thr Leu Gly Cys Gly Ile Glu Met245 250 255 Ser Phe Asp Val Arg Val Gly Val Val Gly Val Glu Ile Ser ThrGly 260 265 270 Glu Val Val Tyr Glu Glu Phe Asn Asp Asn Phe Met Arg SerGly Leu 275 280 285 Glu Ala Val Ile Leu Ser Leu Ser Pro Ala Glu Leu LeuLeu Gly Gln 290 295 300 Pro Leu Ser Gln Gln Thr Glu Lys Phe Leu Val AlaMet Ala Gly Pro 305 310 315 320 Thr Ser Asn Val Arg Val Glu Arg Ala SerLeu Asp Cys Phe Ser Asn 325 330 335 Gly Asn Ala Val Asp Glu Val Ile SerLeu Cys Glu Lys Ile Ser Ala 340 345 350 Gly Asn Leu Glu Asp Asp Lys GluMet Lys Leu Glu Ala Ala Glu Lys 355 360 365 Gly Met Ser Cys Leu Thr ValHis Thr Ile Met Asn Met Pro His Leu 370 375 380 Thr Val Gln Ala Leu AlaLeu Thr Phe Cys His Leu Lys Gln Phe Gly 385 390 395 400 Phe Glu Arg IleLeu Tyr Gln Gly Ala Ser Phe Arg Ser Leu Ser Ser 405 410 415 Asn Thr GluMet Thr Leu Ser Ala Asn Thr Leu Gln Gln Leu Glu Val 420 425 430 Val LysAsn Asn Ser Asp Gly Ser Glu Ser Gly Ser Leu Phe His Asn 435 440 445 MetAsn His Thr Leu Thr Val Tyr Gly Ser Arg Leu Leu Arg His Trp 450 455 460Val Thr His Pro Leu Cys Asp Arg Asn Leu Ile Ser Ala Arg Leu Asp 465 470475 480 Ala Val Ser Glu Ile Ser Ala Cys Met Gly Ser His Ser Ser Ser Gln485 490 495 Leu Ser Ser Glu Leu Val Glu Glu Gly Ser Glu Arg Ala Ile ValSer 500 505 510 Pro Glu Phe Tyr Leu Val Leu Ser Ser Val Leu Thr Ala MetSer Arg 515 520 525 Ser Ser Asp Ile Gln Arg Gly Ile Thr Arg Ile Phe HisArg Thr Ala 530 535 540 Lys Ala Thr Glu Phe Ile Ala Val Met Glu Ala IleLeu Leu Ala Gly 545 550 555 560 Lys Gln Ile Gln Arg Leu Gly Ile Lys GlnAsp Ser Glu Met Arg Ser 565 570 575 Met Gln Ser Ala Thr Val Arg Ser ThrLeu Leu Arg Lys Leu Ile Ser 580 585 590 Val Ile Ser Ser Pro Val Val ValAsp Asn Ala Gly Lys Leu Leu Ser 595 600 605 Ala Leu Asn Lys Glu Ala AlaVal Arg Gly Asp Leu Leu Asp Ile Leu 610 615 620 Ile Thr Ser Ser Asp GlnPhe Pro Glu Leu Ala Glu Ala Arg Gln Ala 625 630 635 640 Val Leu Val IleArg Glu Lys Leu Asp Ser Ser Ile Ala Ser Phe Arg 645 650 655 Lys Lys LeuAla Ile Arg Asn Leu Glu Phe Leu Gln Val Ser Gly Ile 660 665 670 Thr HisLeu Ile Glu Leu Pro Val Asp Ser Lys Val Pro His Asn Trp 675 680 685 ValLys Val Asn Ser Thr Lys Lys Thr Ile Arg Tyr His Pro Pro Glu 690 695 700Ile Val Ala Gly Leu Asp Glu Leu Ala Leu Ala Thr Glu His Leu Ala 705 710715 720 Ile Val Asn Arg Ala Ser Trp Asp Ser Phe Leu Lys Ser Phe Ser Arg725 730 735 Tyr Tyr Thr Asp Phe Lys Ala Ala Val Gln Ala Leu Ala Ala LeuAsp 740 745 750 Cys Leu His Ser Leu Ser Thr Leu Ser Arg Asn Lys Asn TyrVal Arg 755 760 765 Pro Glu Phe Val Asp Asp Cys Glu Pro Val Glu Ile AsnIle Gln Ser 770 775 780 Gly Arg His Pro Val Leu Glu Thr Ile Leu Gln AspAsn Phe Val Pro 785 790 795 800 Asn Asp Thr Ile Leu His Ala Glu Gly GluTyr Cys Gln Ile Ile Thr 805 810 815 Gly Pro Asn Met Gly Gly Lys Ser CysTyr Ile Arg Gln Val Ala Leu 820 825 830 Ile Ser Ile Met Ala Gln Val GlySer Phe Val Pro Ala Ser Phe Ala 835 840 845 Lys Leu His Val Leu Asp GlyVal Phe Thr Arg Met Gly Ala Ser Asp 850 855 860 Ser Ile Gln His Gly ArgSer Thr Phe Leu Glu Glu Leu Ser Glu Ala 865 870 875 880 Ser His Ile IleArg Thr Cys Ser Ser Arg Ser Leu Val Ile Leu Asp 885 890 895 Glu Leu GlyArg Gly Thr Ser Thr His Asp Gly Val Ala Ile Ala Tyr 900 905 910 Ala ThrLeu Gln His Leu Leu Ala Glu Lys Arg Cys Leu Val Leu Phe 915 920 925 ValThr His Tyr Pro Glu Ile Ala Glu Ile Ser Asn Gly Phe Pro Gly 930 935 940Ser Val Gly Thr Tyr His Val Ser Tyr Leu Thr Leu Gln Lys Asp Lys 945 950955 960 Gly Ser Tyr Asp His Asp Asp Val Thr Tyr Leu Tyr Lys Leu Val Arg965 970 975 Gly Leu Cys Ser Arg Ser Phe Gly Phe Lys Val Ala Gln Leu AlaGln 980 985 990 Ile Pro Pro Ser Cys Ile Arg Arg Ala Ile Ser Met Ala AlaLys Leu 995 1000 1005 Glu Ala Glu Val Arg Ala Arg Glu Arg Asn Thr ArgMet Gly Glu Pro 1010 1015 1020 Glu Gly His Glu Glu Pro Arg Gly Ala GluGlu Ser Ile Ser Ala Leu 1025 1030 1035 1040 Gly Asp Leu Phe Ala Asp LeuLys Phe Ala Leu Ser Glu Glu Asp Pro 1045 1050 1055 Trp Lys Ala Phe GluPhe Leu Lys His Ala Trp Lys Ile Ala Gly Lys 1060 1065 1070 Ile Arg LeuLys Pro Thr Cys Ser Phe 1075 1080 20 24 DNA Artificial sequence MSH6specific primer 638 for PCR using cDNA of Arabidopsis thaliana ecotypeColumbia 20 tctctaccag gtgacgaaaa accg 24 21 28 DNA Artificial sequencePrimer S81 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia21 cgtcgccttt agcatcccct tccttcac 28 22 30 DNA Artificial sequence MSH6specific primer S823 for PCR using cDNA of Arabidopsis thaliana ecotypeColumbia 22 gcttggcgca tctaatagaa tcatgacagg 30 23 24 DNA Artificialsequence MSH6 specific primer 637 for PCR using cDNA of Arabidopsisthaliana ecotype Columbia 23 gacagcgtca gttcttcaga atgc 24 24 33 DNAArtificial sequence MSH6 specific primer 1S8 for PCR using cDNA ofArabidopsis thaliana ecotype Columbia 24 atcccgggat gcagcgccagagatcgattt tgt 33 25 27 DNA Artificial sequence MSH6 specific primer S83for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 25cgctatctat ggctgcttcg aattgag 27 26 2188 DNA Arabidopsis thalianaecotype Columbia Clone 43 26 cccgggatgc agcgccagag atcgattttg tctttcttccaaaaacccac ggcggcgact 60 acgaagggtt tggtttccgg cgatgctgct agcggcgggggcggcagcgg aggaccacga 120 tttaatgtga aggaagggga tgctaaaggc gacgcttctgtacgttttgc tgtttcgaaa 180 tctgtcgatg aggttagagg aacggatact ccaccggagaaggttccgcg tcgtgtcctg 240 ccgtctggat ttaagccggc tgaatccgcc ggtgatgcttcgtccctgtt ctccaatatt 300 atgcataagt ttgtaaaagt cgatgatcga gattgttctggagagaggag ccgagaagat 360 gttgttccgc tgaatgattc atctctatgt atgaaggctaatgatgttat tcctcaattt 420 cgttccaata atggtaaaac tcaagaaaga aaccatgcttttagtttcag tgggagagct 480 gaacttagat cagtagaaga tataggagta gatggcgatgttcctggtcc agaaacacca 540 gggatgcgtc cacgtgcttc tcgcttgaag cgagttctggaggatgaaat gacttttaag 600 gaggataagg ttcctgtatt ggactctaac aaaaggctgaaaatgctcca ggatccggtt 660 tgtggagaga agaaagaagt aaacgaagga accaaatttgaatggcttga gtcttctcga 720 atcagggatg ccaatagaag acgtcctgat gatcccctttacgatagaaa gaccttacac 780 ataccacctg atgttttcaa gaaaatgtct gcatcacaaaagcaatattg gagtgttaag 840 agtgaatata tggacattgt gcttttcttt aaagtggggaaattttatga gctgtatgag 900 ctagatgcgg aattaggtca caaggagctt gactggaagatgaccatgag tggtgtggga 960 aaatgcagac aggttggtat ctctgaaagt gggatagatgaggcagtgca aaagctatta 1020 gctcgtggat ataaagttgg acgaatcgag cagctagaaacatctgacca agcaaaagcc 1080 agaggtgcta atactataat tccaaggaag ctagttcaggtattaactcc atcaacagca 1140 agcgagggaa acatcgggcc tgatgccgtc catcttcttgctataaaaga gatcaaaatg 1200 gagctacaaa agtgttcaac tgtgtatgga tttgcttttgttgactgtgc tgccttgagg 1260 ttttgggttg ggtccatcag cgatgatgca tcatgtgctgctcttggagc gttattgatg 1320 caggtttctc caaaggaagt gttatatgac agtaaagggctatcaagaga agcacaaaag 1380 gctctaagga aatatacgtt gacagggtct acggcggtacagttggctcc agtaccacaa 1440 gtaatggggg atacagatgc tgctggagtt agaaatataatagaatctaa cggatacttt 1500 aaaggttctt ctgaatcatg gaactgtgct gttgatggtctaaatgaatg tgatgttgcc 1560 cttagtgctc ttggagagct aattaatcat ctgtctaggctaaagctaga agatgtactt 1620 aagcatgggg atatttttcc ataccaagtt tacaggggttgtctcagaat tgatggccag 1680 acgatggtaa atcttgagat atttaacaat agctgtgatggtggtccttc agggaccttg 1740 tacaaatatc ttgataactg tgttagtcca actggtaagcgactcttaag gaattggatc 1800 tgccatccac tcaaagatgt agaaagcatc aataaacggcttgatgtagt tgaagaattc 1860 acggcaaact cagaaagtat gcaaatcact ggccagtatctccacaaact tccagactta 1920 gaaagactgc tcggacgcat caagtctagc gttcgatcatcagcctctgt gttgcctgct 1980 cttctgggga aaaaagtgct gaaacaacga gttaaagcatttgggcaaat tgtgaaaggg 2040 ttcagaagtg gaattgatct gttgttggct ctacagaaggaatcaaatat gatgagtttg 2100 ctttataaac tctgtaaact tcctatatta gtaggaaaaagcgggctaga gttatttctt 2160 tctcaattcg aagcagccat agatagcg 2188 27 1385DNA Arabidopsis thaliana ecotype Columbia Clone 62 27 catcagcctctgtgttgcct gctcttctgg ggaaaaaagt gctgaaacaa cgagttaaag 60 catttgggcaaattgtgaaa gggttcagaa gtggaattga tctgttgttg gctctacaga 120 aggaatcaaatatgatgagt ttgctttata aactctgtaa acttcctata ttagtaggaa 180 aaagcgggctagagttattt ctttctcaat tcgaagcagc catagatagc gactttccaa 240 attatcagaaccaagatgtg acagatgaaa acgctgaaac tctcacaata cttatcgaac 300 tttttatcgaaagagcaact caatggtctg aggtcattca caccataagc tgcctagatg 360 tcctgagatcttttgcaatc gcagcaagtc tctctgctgg aagcatggcc aggcctgtta 420 tttttcccgaatcagaagct acagatcaga atcagaaaac aaaagggcca atacttaaaa 480 tccaaggactatggcatcca tttgcagttg cagccgatgg tcaattgcct gttccgaatg 540 atatactccttggcgaggct agaagaagca gtggcagcat tcatcctcgg tcattgttac 600 tgacgggaccaaacatgggc ggaaaatcaa ctcttcttcg tgcaacatgt ctggccgtta 660 tctttgcccaacttggctgc tacgtgccgt gtgagtcttg cgaaatctcc ctcgtggata 720 ctatcttcacaaggcttggc gcatctgata gaatcatgac aggagagagt acctttttgg 780 tagaatgcactgagacagcg tcagttcttc agaatgcaac tcaggattca ctagtaatcc 840 ttgacgaactgggcagagga actagtactt tcgatggata cgccattgca tactcggttt 900 ttcgtcacctggtagagaaa gttcaatgtc ggatgctctt tgcaacacat taccaccctc 960 tcaccaaggaattcgcgtct cacccacgtg tcacctcgaa acacatggct tgcgcattca 1020 aatcaagatctgattatcaa ccacgtggtt gtgatcaaga cctagtgttc ttgtaccgtt 1080 taaccgagggagcttgtcct gagagctacg gacttcaagt ggcactcatg gctggaatac 1140 caaaccaagtggttgaaaca gcatcaggtg ctgctcaagc catgaagaga tcaattgggg 1200 aaaacttcaagtcaagtgag ctaagatctg agttctcaag tctgcatgaa gactggctca 1260 agtcattggtgggtatttct cgagtcgccc acaacaatgc ccccattggc gaagatgact 1320 acgacactttgttttgctta tggcatgaga tcaaatcctc ttactgtgtt cccaaataac 1380 ccggg 138528 34 DNA Artificial sequence MSH6 specific primer 2S8 for PCR usingcDNA of Arabidopsis thaliana ecotype Columbia 28 atcccgggtt atttgggaacacagtaagag gatt 34 29 27 DNA Artificial sequence MSH6 specific primerS82 for PCR using cDNA of Arabidopsis thaliana ecotype Columbia 29gcgttcgatc atcagcctct gtgttgc 27 30 3606 DNA Arabidopsis thalianaecotype Columbia CDS (142)....(3468) AtMSH6 full-length cDNA and deducedsequence of the encoded polypeptide 30 aaaagttgag ccctgaggag tatcgtttccgccatttcta cgacgcaagg cgaaaatttt 60 tggcgccaat ctttcccccc tttcgaattctctcagctca aaacatcgtt tctctctcac 120 tctctctcac aattccaaaa a atg cag cgccag aga tcg att ttg tct ttc 171 Met Gln Arg Gln Arg Ser Ile Leu Ser Phe1 5 10 ttc caa aaa ccc acc gcg gcg act acg aag ggt ttg gtt tcc ggc gat219 Phe Gln Lys Pro Thr Ala Ala Thr Thr Lys Gly Leu Val Ser Gly Asp 1520 25 gct gct agc ggc ggg ggc ggc agc gga gga cca cga ttt aat gtg aag267 Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Pro Arg Phe Asn Val Arg 3035 40 gaa ggg gat gct aaa ggc gac gct tct gta cgt ttt gct gtt tcg aaa315 Glu Gly Asp Ala Lys Gly Asp Ala Ser Val Arg Phe Ala Val Ser Lys 4550 55 tct gtc gat gag gtt aga gga acg gat act cca ccg gag aag gtt ccg363 Ser Val Asp Glu Val Arg Gly Thr Asp Thr Pro Pro Glu Lys Val Pro 6065 70 cgt cgt gtc ctg ccg tct gga ttt aag ccg gct gaa tcc gcc gst gat411 Arg Arg Val Leu Pro Ser Gly Phe Lys Pro Ala Glu Ser Ala Gly Asp 7580 85 90 gct tcg tcc ctg ttc tcc aat att atg cat aag ttt gta aaa gtc gat459 Ala Ser Ser Leu Phe Ser Asn Ile Met His Lys Phe Val Lys Val Asp 95100 105 gat cga gat tgt tct gga gag agg agc cga gaa gat gtt gtt ccg ctg507 Asp Arg Asp Cys Ser Gly Glu Arg Ser Arg Glu Asp Val Val Pro Leu 110115 120 aat gat tca tct cta tgt atg aag gct aat gat gtt att cct caa ttt555 Asn Asp Ser Ser Leu Cys Met Lys Ala Asn Asp Val Ile Pro Gln Phe 125130 135 cgt tcc aat aat ggt aaa act caa gaa aga aac cat gct ttt agt ttc603 Arg Ser Asn Asn Gly Lys Thr Gln Glu Arg Asn His Ala Phe Ser Phe 140145 150 agt ggg aga gct gaa ctt aga tca gta gaa gat ata gga gta gat ggc651 Ser Gly Arg Ala Glu Leu Arg Ser Val Glu Asp Ile Gly Val Asp Gly 155160 165 170 gat gtt cct ggt cca gaa aca cca ggg atg cgt cca cgt gct tctcgc 699 Asp Val Pro Gly Pro Glu Thr Pro Gly Met Arg Pro Arg Ala Ser Arg175 180 185 ttg aag cga gtt ctg gag gat gaa atg act ttt aag gag gat aaggtt 747 Leu Lys Arg Val Leu Glu Asp Glu Met Thr Phe Lys Glu Asp Lys Val190 195 200 cct gta ttg gac tct aac aaa agg ctg aaa atg ctc cag gat ccggtt 795 Pro Val Leu Asp Ser Asn Lys Arg Leu Lys Met Leu Gln Asp Pro Val205 210 215 tgt gga gag aag aaa gaa gta aac gaa gga acc aaa ttt gaa tggctt 843 Cys Gly Glu Lys Lys Glu Val Asn Glu Gly Thr Lys Phe Glu Trp Leu220 225 230 gag tct tct cga atc agg gat gcc aat aga aga cgt cct gat gatccc 891 Glu Ser Ser Arg Ile Arg Asp Ala Asn Arg Arg Arg Pro Asp Asp Pro235 240 245 250 ctt tac gat aga aag acc tta cac ata cca cct gat gtt ttcaag aaa 939 Leu Tyr Asp Arg Lys Thr Leu His Ile Pro Pro Asp Val Phe LysLys 255 260 265 atg tct gca tca caa aag caa tat tgg agt gtt aag agt gaatat atg 987 Met Ser Ala Ser Gln Lys Gln Tyr Trp Ser Val Lys Ser Glu TyrMet 270 275 280 gac att gtg ctt ttc ttt aaa gtg ggg aaa ttt tat gag ctgtat gag 1035 Asp Ile Val Leu Phe Phe Lys Val Gly Lys Phe Tyr Glu Leu TyrGlu 285 290 295 cta gat gcg gaa tta ggt cac aag gag ctt gac tgg aag atgacc atg 1083 Leu Asp Ala Glu Leu Gly His Lys Glu Leu Asp Trp Lys Met ThrMet 300 305 310 agt ggt gtg gga aaa tgc aga cag gtt ggt atc tct gaa agtggg ata 1131 Ser Gly Val Gly Lys Cys Arg Gln Val Gly Ile Ser Glu Ser GlyIle 315 320 325 330 gat gag gca gtg caa aag cta tta gct cgt gga tat aaagtt gga cga 1179 Asp Glu Ala Val Gln Lys Leu Leu Ala Arg Gly Tyr Lys ValGly Arg 335 340 345 atc gag cag cta gaa aca tct gac caa gca aaa gcc agaggt gct aat 1227 Ile Glu Gln Leu Glu Thr Ser Asp Gln Ala Lys Ala Arg GlyAla Asn 350 355 360 act ata att cca agg aag cta gtt cag gta tta act ccatca aca gca 1275 Thr Ile Ile Pro Arg Lys Leu Val Gln Val Leu Thr Pro SerThr Ala 365 370 375 agc gag gga aac atc ggg cct gat gcc gtc cat ctt cttgct ata aaa 1323 Ser Glu Gly Asn Ile Gly Pro Asp Ala Val His Leu Leu AlaIle Lys 380 385 390 gag atc aaa atg gag cta caa aag tgt tca act gtg tatgga ttt gct 1371 Glu Ile Lys Met Glu Leu Gln Lys Cys Ser Thr Val Tyr GlyPhe Ala 395 400 405 410 ttt gtt gac tgt gct gcc ttg agg ttt tgg gtt gggtcc atc agc gat 1419 Phe Val Asp Cys Ala Ala Leu Arg Phe Trp Val Gly SerIle Ser Asp 415 420 425 gat gca tca tgt gct gct ctt gga gcg tta ttg atgcag gtt tct cca 1467 Asp Ala Ser Cys Ala Ala Leu Gly Ala Leu Leu Met GlnVal Ser Pro 430 435 440 aag gaa gtg tta tat gac agt aaa ggg cta tca agagaa gca caa aag 1515 Lys Glu Val Leu Tyr Asp Ser Lys Gly Leu Ser Arg GluAla Gln Lys 445 450 455 gct cta agg aaa tat acg ttg aca ggg tct acg gcggta cag ttg gct 1563 Ala Leu Arg Lys Tyr Thr Leu Thr Gly Ser Thr Ala ValGln Leu Ala 460 465 470 cca gta cca caa gta atg ggg gat aca gat gct gctgga gtt aga aat 1611 Pro Val Pro Gln Val Met Gly Asp Thr Asp Ala Ala GlyVal Arg Asn 475 480 485 490 ata ata gaa tct aac gga tac ttt aaa ggt tcttct gaa tca tgg aac 1659 Ile Ile Glu Ser Asn Gly Tyr Phe Lys Gly Ser SerGlu Ser Trp Asn 495 500 505 tgt gct gtt gat ggt cta aat gaa tgt gat gttgcc ctt agt gct ctt 1707 Cys Ala Val Asp Gly Leu Asn Glu Cys Asp Val AlaLeu Ser Ala Leu 510 515 520 gga gag cta att aat cat ctg tct agg cta aagcta gaa gat gta ctt 1755 Gly Glu Leu Ile Asn His Leu Ser Arg Leu Lys LeuGlu Asp Val Leu 525 530 535 aag cat ggg gat att ttt cca tac caa gtt tacagg ggt tgt ctc aga 1803 Lys His Gly Asp Ile Phe Pro Tyr Gln Val Tyr ArgGly Cys Leu Arg 540 545 550 att gat ggc cag acg atg gta aat ctt gag atattt aac aat agc tgt 1851 Ile Asp Gly Gln Thr Met Val Asn Leu Glu Ile PheAsn Asn Ser Cys 555 560 565 570 gat ggt ggt cct tca ggg acc ttg tac aaatat ctt gat aac tgt gtt 1899 Asp Gly Gly Pro Ser Gly Thr Leu Tyr Lys TyrLeu Asp Asn Cys Val 575 580 585 agt cca act ggt aag cga ctc tta agg aattgg atc tgc cat cca ctc 1947 Ser Pro Thr Gly Lys Arg Leu Leu Arg Asn TrpIle Cys His Pro Leu 590 595 600 aaa gat gta gaa agc atc aat aaa cgg cttgat gta gtt gaa gaa ttc 1995 Lys Asp Val Glu Ser Ile Asn Lys Arg Leu AspVal Val Glu Glu Phe 605 610 615 acg gca aac tca gaa agt atg caa atc actggc cag tat ctc cac aaa 2043 Thr Ala Asn Ser Glu Ser Met Gln Ile Thr GlyGln Tyr Leu His Lys 620 625 630 ctt cca gac tta gaa aga ctg ctc gga cgcatc aag tct agc gtt cga 2091 Leu Pro Asp Leu Glu Arg Leu Leu Gly Arg IleLys Ser Ser Val Arg 635 640 645 650 tca tca gcc tct gtg ttg cct gct cttctg ggg aaa aaa gtg ctg aaa 2139 Ser Ser Ala Ser Val Leu Pro Ala Leu LeuGly Lys Lys Val Leu Lys 655 660 665 caa cga gtt aaa gca ttt ggg caa attgtg aaa ggg ttc aga agt gga 2187 Gln Arg Val Lys Ala Phe Gly Gln Ile ValLys Gly Phe Arg Ser Gly 670 675 680 att gat ctg ttg ttg gct cta cag aaggaa tca aat atg atg agt ttg 2235 Ile Asp Leu Leu Leu Ala Leu Gln Lys GluSer Asn Met Met Ser Leu 685 690 695 ctt tat aaa ctc tgt aaa ctt cct atatta gta gga aaa agc ggg cta 2283 Leu Tyr Lys Leu Cys Lys Leu Pro Ile LeuVal Gly Lys Ser Gly Leu 700 705 710 gag tta ttt ctt tct caa ttc gaa gcagcc ata gat agc gac ttt cca 2331 Glu Leu Phe Leu Ser Gln Phe Glu Ala AlaIle Asp Ser Asp Phe Pro 715 720 725 730 aat tat cag aac caa gat gtg acagat gaa aac gct gaa act ctc aca 2379 Asn Tyr Gln Asn Gln Asp Val Thr AspGlu Asn Ala Glu Thr Leu Thr 735 740 745 ata ctt atc gaa ctt ttt atc gaaaga gca act caa tgg tct gag gtc 2427 Ile Leu Ile Glu Leu Phe Ile Glu ArgAla Thr Gln Trp Ser Glu Val 750 755 760 att cac acc ata agc tgc cta gatgtc ctg aga tct ttt gca atc gca 2475 Ile His Thr Ile Ser Cys Leu Asp ValLeu Arg Ser Phe Ala Ile Ala 765 770 775 gca agt ctc tct gct gga agc atggcc agg cct gtt att ttt ccc gaa 2523 Ala Ser Leu Ser Ala Gly Ser Met AlaArg Pro Val Ile Phe Pro Glu 780 785 790 tca gaa gct aca gat cag aat cagaaa aca aaa ggg cca ata ctt aaa 2571 Ser Glu Ala Thr Asp Gln Asn Gln LysThr Lys Gly Pro Ile Leu Lys 795 800 805 810 atc caa gga cta tgg cat ccattt gca gtt gca gcc gat ggt caa ttg 2619 Ile Gln Gly Leu Trp His Pro PheAla Val Ala Ala Asp Gly Gln Leu 815 820 825 cct gtt ccg aat gat ata ctcctt ggc gag gct aga aga agc agt ggc 2667 Pro Val Pro Asn Asp Ile Leu LeuGly Glu Ala Arg Arg Ser Ser Gly 830 835 840 agc att cat cct cgg tca ttgtta ctg acg gga cca aac atg ggc gga 2715 Ser Ile His Pro Arg Ser Leu LeuLeu Thr Gly Pro Asn Met Gly Gly 845 850 855 aaa tca act ctt ctt cgt gcaaca tgt ctg gcc gtt atc ttt gcc caa 2763 Lys Ser Thr Leu Leu Arg Ala ThrCys Leu Ala Val Ile Phe Ala Gln 860 865 870 ctt ggc tgc tac gtg ccg tgtgag tct tgc gaa atc tcc ctc gtg gat 2811 Leu Gly Cys Tyr Val Pro Cys GluSer Cys Glu Ile Ser Leu Val Asp 875 880 885 890 act atc ttc aca agg cttggc gca tct gat aga atc atg aca gga gag 2859 Thr Ile Phe Thr Arg Leu GlyAla Ser Asp Arg Ile Met Thr Gly Glu 895 900 905 agt acc ttt ttg gta gaatgc act gag aca gcg tca gtt ctt cag aat 2907 Ser Thr Phe Leu Val Glu CysThr Glu Thr Ala Ser Val Leu Gln Asn 910 915 920 gca act cag gat tca ctagta atc ctt gac gaa ctg ggc aga gga act 2955 Ala Thr Gln Asp Ser Leu ValIle Leu Asp Glu Leu Gly Arg Gly Thr 925 930 935 agt act ttc gat gga tacgcc att gca tac tcg gtt ttt cgt cac ctg 3003 Ser Thr Phe Asp Gly Tyr AlaIle Ala Tyr Ser Val Phe Arg His Leu 940 945 950 gta gag aaa gtt caa tgtcgg atg ctc ttt gca aca cat tac cac cct 3051 Val Glu Lys Val Gln Cys ArgMet Leu Phe Ala Thr His Tyr His Pro 955 960 965 970 ctc acc aag gaa ttcgcg tct cac cca cgt gtc acc tcg aaa cac atg 3099 Leu Thr Lys Glu Phe AlaSer His Pro Arg Val Thr Ser Lys His Met 975 980 985 gct tgc gca ttc aaatca aga tct gat tat caa cca cgt ggt tgt gat 3147 Ala Cys Ala Phe Lys SerArg Ser Asp Tyr Gln Pro Arg Gly Cys Asp 990 995 1000 caa gac cta gtg ttcttg tac cgt tta acc gag gga gct tgt cct gag 3195 Gln Asp Leu Val Phe LeuTyr Arg Leu Thr Glu Gly Ala Cys Pro Glu 1005 1010 1015 agc tac gga cttcaa gtg gca ctc atg gct gga ata cca aac caa gtg 3243 Ser Tyr Gly Leu GlnVal Ala Leu Met Ala Gly Ile Pro Asn Gln Val 1020 1025 1030 gtt gaa acagca tca ggt gct gct caa gcc atg aag aga tca att ggg 3291 Val Glu Thr AlaSer Gly Ala Ala Gln Ala Met Lys Arg Ser Ile Gly 1035 1040 1045 1050 ggaaac ttc aag tca agt gag cta aga tct gag ttc tca agt ctg cat 3339 Glu AsnPhe Lys Ser Ser Glu Leu Arg Ser Glu Phe Ser Ser Leu His 1055 1060 1065gaa gac tgg ctc aag tca ttg gtg ggt att tct cga gtc gcc cac aac 3387 GluAsp Trp Leu Lys Ser Leu Val Gly Ile Ser Arg Val Ala His Asn 1070 10751080 aat gcc ccc att ggc gaa gat gac tac gac act ttg ttt tgc tta tgg3435 Asn Ala Pro Ile Gly Glu Asp Asp Tyr Asp Thr Leu Phe Cys Leu Trp1085 1090 1095 cat gag atc aaa tcc tct tac tgt gtt ccc aaa taaatggcta3478 His Glu Ile Lys Ser Ser Tyr Cys Val Pro Lys 1100 1105 tgacataacactatctgaag ctcgttaagt cttttgcctc tctgatgttt attcctctta 3538 aaaaatgcttatatatcaaa aaattgtttc ctcgattaaa aaaaaaaaaa aaaaaaaaaa 3598 aaaaaaaa3606 31 1109 PRT Arabidopsis thaliana ecotype Columbia Polypeptide MSH631 Met Gln Arg Gln Arg Ser Ile Leu Ser Phe Phe Gln Lys Pro Thr Ala 1 510 15 Ala Thr Thr Lys Gly Leu Val Ser Gly Asp Ala Ala Ser Gly Gly Gly 2025 30 Gly Ser Gly Gly Pro Arg Phe Asn Val Arg Glu Gly Asp Ala Lys Gly 3540 45 Asp Ala Ser Val Arg Phe Ala Val Ser Lys Ser Val Asp Glu Val Arg 5055 60 Gly Thr Asp Thr Pro Pro Glu Lys Val Pro Arg Arg Val Leu Pro Ser 6570 75 80 Gly Phe Lys Pro Ala Glu Ser Ala Gly Asp Ala Ser Ser Leu Phe Ser85 90 95 Asn Ile Met His Lys Phe Val Lys Val Asp Asp Arg Asp Cys Ser Gly100 105 110 Glu Arg Ser Arg Glu Asp Val Val Pro Leu Asn Asp Ser Ser LeuCys 115 120 125 Met Lys Ala Asn Asp Val Ile Pro Gln Phe Arg Ser Asn AsnGly Lys 130 135 140 Thr Gln Glu Arg Asn His Ala Phe Ser Phe Ser Gly ArgAla Glu Leu 145 150 155 160 Arg Ser Val Glu Asp Ile Gly Val Asp Gly AspVal Pro Gly Pro Glu 165 170 175 Thr Pro Gly Met Arg Pro Arg Ala Ser ArgLeu Lys Arg Val Leu Glu 180 185 190 Asp Glu Met Thr Phe Lys Glu Asp LysVal Pro Val Leu Asp Ser Asn 195 200 205 Lys Arg Leu Lys Met Leu Gln AspPro Val Cys Gly Glu Lys Lys Glu 210 215 220 Val Asn Glu Gly Thr Lys PheGlu Trp Leu Glu Ser Ser Arg Ile Arg 225 230 235 240 Asp Ala Asn Arg ArgArg Pro Asp Asp Pro Leu Tyr Asp Arg Lys Thr 245 250 255 Leu His Ile ProPro Asp Val Phe Lys Lys Met Ser Ala Ser Gln Lys 260 265 270 Gln Tyr TrpSer Val Lys Ser Glu Tyr Met Asp Ile Val Leu Phe Phe 275 280 285 Lys ValGly Lys Phe Tyr Glu Leu Tyr Glu Leu Asp Ala Glu Leu Gly 290 295 300 HisLys Glu Leu Asp Trp Lys Met Thr Met Ser Gly Val Gly Lys Cys 305 310 315320 Arg Gln Val Gly Ile Ser Glu Ser Gly Ile Asp Glu Ala Val Gln Lys 325330 335 Leu Leu Ala Arg Gly Tyr Lys Val Gly Arg Ile Glu Gln Leu Glu Thr340 345 350 Ser Asp Gln Ala Lys Ala Arg Gly Ala Asn Thr Ile Ile Pro ArgLys 355 360 365 Leu Val Gln Val Leu Thr Pro Ser Thr Ala Ser Glu Gly AsnIle Gly 370 375 380 Pro Asp Ala Val His Leu Leu Ala Ile Lys Glu Ile LysMet Glu Leu 385 390 395 400 Gln Lys Cys Ser Thr Val Tyr Gly Phe Ala PheVal Asp Cys Ala Ala 405 410 415 Leu Arg Phe Trp Val Gly Ser Ile Ser AspAsp Ala Ser Cys Ala Ala 420 425 430 Leu Gly Ala Leu Leu Met Gln Val SerPro Lys Glu Val Leu Tyr Asp 435 440 445 Ser Lys Gly Leu Ser Arg Glu AlaGln Lys Ala Leu Arg Lys Tyr Thr 450 455 460 Leu Thr Gly Ser Thr Ala ValGln Leu Ala Pro Val Pro Gln Val Met 465 470 475 480 Gly Asp Thr Asp AlaAla Gly Val Arg Asn Ile Ile Glu Ser Asn Gly 485 490 495 Tyr Phe Lys GlySer Ser Glu Ser Trp Asn Cys Ala Val Asp Gly Leu 500 505 510 Asn Glu CysAsp Val Ala Leu Ser Ala Leu Gly Glu Leu Ile Asn His 515 520 525 Leu SerArg Leu Lys Leu Glu Asp Val Leu Lys His Gly Asp Ile Phe 530 535 540 ProTyr Gln Val Tyr Arg Gly Cys Leu Arg Ile Asp Gly Gln Thr Met 545 550 555560 Val Asn Leu Glu Ile Phe Asn Asn Ser Cys Asp Gly Gly Pro Ser Gly 565570 575 Thr Leu Tyr Lys Tyr Leu Asp Asn Cys Val Ser Pro Thr Gly Lys Arg580 585 590 Leu Leu Arg Asn Trp Ile Cys His Pro Leu Lys Asp Val Glu SerIle 595 600 605 Asn Lys Arg Leu Asp Val Val Glu Glu Phe Thr Ala Asn SerGlu Ser 610 615 620 Met Gln Ile Thr Gly Gln Tyr Leu His Lys Leu Pro AspLeu Glu Arg 625 630 635 640 Leu Leu Gly Arg Ile Lys Ser Ser Val Arg SerSer Ala Ser Val Leu 645 650 655 Pro Ala Leu Leu Gly Lys Lys Val Leu LysGln Arg Val Lys Ala Phe 660 665 670 Gly Gln Ile Val Lys Gly Phe Arg SerGly Ile Asp Leu Leu Leu Ala 675 680 685 Leu Gln Lys Glu Ser Asn Met MetSer Leu Leu Tyr Lys Leu Cys Lys 690 695 700 Leu Pro Ile Leu Val Gly LysSer Gly Leu Glu Leu Phe Leu Ser Gln 705 710 715 720 Phe Glu Ala Ala IleAsp Ser Asp Phe Pro Asn Tyr Gln Asn Gln Asp 725 730 735 Val Thr Asp GluAsn Ala Glu Thr Leu Thr Ile Leu Ile Glu Leu Phe 740 745 750 Ile Glu ArgAla Thr Gln Trp Ser Glu Val Ile His Thr Ile Ser Cys 755 760 765 Leu AspVal Leu Arg Ser Phe Ala Ile Ala Ala Ser Leu Ser Ala Gly 770 775 780 SerMet Ala Arg Pro Val Ile Phe Pro Glu Ser Glu Ala Thr Asp Gln 785 790 795800 Asn Gln Lys Thr Lys Gly Pro Ile Leu Lys Ile Gln Gly Leu Trp His 805810 815 Pro Phe Ala Val Ala Ala Asp Gly Gln Leu Pro Val Pro Asn Asp Ile820 825 830 Leu Leu Gly Glu Ala Arg Arg Ser Ser Gly Ser Ile His Pro ArgSer 835 840 845 Leu Leu Leu Thr Gly Pro Asn Met Gly Gly Lys Ser Thr LeuLeu Arg 850 855 860 Ala Thr Cys Leu Ala Val Ile Phe Ala Gln Leu Gly CysTyr Val Pro 865 870 875 880 Cys Glu Ser Cys Glu Ile Ser Leu Val Asp ThrIle Phe Thr Arg Leu 885 890 895 Gly Ala Ser Asp Arg Ile Met Thr Gly GluSer Thr Phe Leu Val Glu 900 905 910 Cys Thr Glu Thr Ala Ser Val Leu GlnAsn Ala Thr Gln Asp Ser Leu 915 920 925 Val Ile Leu Asp Glu Leu Gly ArgGly Thr Ser Thr Phe Asp Gly Tyr 930 935 940 Ala Ile Ala Tyr Ser Val PheArg His Leu Val Glu Lys Val Gln Cys 945 950 955 960 Arg Met Leu Phe AlaThr His Tyr His Pro Leu Thr Lys Glu Phe Ala 965 970 975 Ser His Pro ArgVal Thr Ser Lys His Met Ala Cys Ala Phe Lys Ser 980 985 990 Arg Ser AspTyr Gln Pro Arg Gly Cys Asp Gln Asp Leu Val Phe Leu 995 1000 1005 TyrArg Leu Thr Glu Gly Ala Cys Pro Glu Ser Tyr Gly Leu Gln Val 1010 10151020 Ala Leu Met Ala Gly Ile Pro Asn Gln Val Val Glu Thr Ala Ser Gly1025 1030 1035 1040 Ala Ala Gln Ala Met Lys Arg Ser Ile Gly Glu Asn PheLys Ser Ser 1045 1050 1055 Glu Leu Arg Ser Glu Phe Ser Ser Leu His GluAsp Trp Leu Lys Ser 1060 1065 1070 Leu Val Gly Ile Ser Arg Val Ala HisAsn Asn Ala Pro Ile Gly Glu 1075 1080 1085 Asp Asp Tyr Asp Thr Leu PheCys Leu Trp His Glu Ile Lys Ser Ser 1090 1095 1100 Tyr Cys Val Pro Lys1105 32 24 DNA Artificial sequence Forward primer for PCR amplificationof ATHGENEA microsatellite 32 accatgcata gcttaaactt cttg 24 33 22 DNAArtificial sequence Reverse primer for PCR amplification of ATHGENEAmicrosatellite 33 acataaccac aaataggggt gc 22 34 18 DNA Artificialsequence Forward primer DMCIN-A for PCR on genomic DNA of Arabidopsisthaliana ssp. Landsberg erecta “Ler” 34 gaagcgatat tgttcgtg 18 35 18 DNAArtificial sequence Reverse primer DMCIN-B for PCR on genomic DNA ofArabidopsis thaliana ssp. Landsberg erecta “Ler” 35 agattgcgag aacattcc18 36 31 DNA Artificial sequence Forward primer DMCIN-1 for PCR ongenomic DNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 36acgcgtcgac tcagctatga gattactcgt g 31 37 29 DNA Artificial sequenceReverse primer DMCIN-2 for PCR on genomic DNA of Arabidopsis thalianassp. Landsberg erecta “Ler” 37 gctctagatt tctcgctcta agactctct 29 38 32DNA Artificial sequence Forward primer DMCIN-3 for PCR on genomic DNA ofArabidopsis thaliana ssp. Landsberg erecta “Ler” 38 gctctagagcttctcttaag taagtgattg at 32 39 48 DNA Artificial sequence Reverse primerDMCIN-4 for PCR on genomic DNA of Arabidopsis thaliana ssp. Landsbergerecta “Ler” 39 tcccccgggc tcgagagatc tccatggttt cttcagctct atgaatcc 4840 26 DNA Artificial sequence Forward primer DMC1a for PCR on genomicDNA of Arabidopsis thaliana ssp. Landsberg erecta “Ler” 40 acgcgtcgacgaattcgcaa gtgggg 26 41 38 DNA Artificial sequence Reverse primer DMC1bfor PCR on genomic DNA of Arabidopsis thaliana ssp. Landsberg erecta“Ler” 41 tccatggaga tctcccgggt accgatttgc ttcgaggg 38 42 20 DNAArtificial sequence Forward primer for PCR amplification of ATEAT1 SSLPmarker in Arabidopsis thaliana subspecies 42 gccactgcgt gaatgatatg 20 4322 DNA Artificial sequence Reverse primer for PCR amplification ofATEAT1 SSLP marker in Arabidopsis thaliana subspecies 43 cgaacagccaacattaattc cc 22 44 18 DNA Artificial sequence Forward primer for PCRamplification of NGA63 SSLP marker in Arabidopsis thaliana subspecies 44aaccaaggca cagaagcg 18 45 18 DNA Artificial sequence Reverse primer forPCR amplification of NGA63 SSLP marker in Arabidopsis thalianasubspecies 45 acccaagtga tcgccacc 18 46 21 DNA Artificial sequenceForward primer for PCR amplification of NGA248 SSLP marker inArabidopsis thaliana subspecies 46 taccgaacca aaacacaaag g 21 47 22 DNAArtificial sequence Reverse primer for PCR amplification of NGA248 SSLPmarker in Arabidopsis thaliana subspecies 47 tctgtatctc ggtgaattct cc 2248 22 DNA Artificial sequence Forward primer for PCR amplification ofNGA128 SSLP marker in Arabidopsis thaliana subspecies 48 ggtctgttgatgtcgtaagt cg 22 49 22 DNA Artificial sequence Reverse primer for PCRamplification of NGA128 SSLP marker in Arabidopsis thaliana subspecies49 atcttgaaac ctttagggag gg 22 50 22 DNA Artificial sequence Forwardprimer for PCR amplification of NGA280 SSLP marker in Arabidopsisthaliana subspecies 50 ctgatctcac ggacaatagt gc 22 51 20 DNA Artificialsequence Reverse primer for PCR amplification of NGA280 SSLP marker inArabidopsis thaliana subspecies 51 ggctccataa aaagtgcacc 20 52 21 DNAArtificial sequence Forward primer for PCR amplification of NGA111 SSLPmarker in Arabidopsis thaliana subspecies 52 ctccagttgg aagctaaagg g 2153 21 DNA Artificial sequence Reverse primer for PCR amplification ofNGA111 SSLP marker in Arabidopsis thaliana subspecies 53 tgttttttaggacaaatggc g 21 54 20 DNA Artificial sequence Forward primer for PCRamplification of NGA168 SSLP marker in Arabidopsis thaliana subspecies54 ccttcacatc caaaacccac 20 55 20 DNA Artificial sequence Reverse primerfor PCR amplification of NGA168 SSLP marker in Arabidopsis thalianasubspecies 55 gcacataccc acaaccagaa 20 56 20 DNA Artificial sequenceForward primer for PCR amplification of NGA1126 SSLP marker inArabidopsis thaliana subspecies 56 cgctacgctt ttcggtaaag 20 57 20 DNAArtificial sequence Reverse primer for PCR amplification of NGA1126 SSLPmarker in Arabidopsis thaliana subspecies 57 gcacagtcca agtcacaacc 20 5820 DNA Artificial sequence Forward primer for PCR amplification ofNGA361 SSLP marker in Arabidopsis thaliana subspecies 58 aaagagatgagaatttggac 20 59 23 DNA Artificial sequence Reverse primer for PCRamplification of NGA361 SSLP marker in Arabidopsis thaliana subspecies59 acatatcaat atattaaagt agc 23 60 18 DNA Artificial sequence Forwardprimer for PCR amplification of NGA168 SSLP marker in Arabidopsisthaliana subspecies 60 tcgtctactg cactgccg 18 61 22 DNA Artificialsequence Reverse primer for PCR amplification of NGA168 SSLP marker inArabidopsis thaliana subspecies 61 gaggacatgt ataggagcct cg 22 62 20 DNAArtificial sequence Forward primer for PCR amplification of AthBIO2 SSLPmarker in Arabidopsis thaliana subspecies 62 tgacctcctc ttccatggag 20 6322 DNA Artificial sequence Reverse primer for PCR amplification ofAthBIO2 SSLP marker in Arabidopsis thaliana subspecies 63 ttaacagaaacccaaagctt tc 22 64 21 DNA Artificial sequence Forward primer for PCRamplification of AthUBIQUE SSLP marker in Arabidopsis thalianasubspecies 64 aggcaaatgt ccatttcatt g 21 65 20 DNA Artificial sequenceReverse primer for PCR amplification of AthUBIQUE SSLP marker inArabidopsis thaliana subspecies 65 acgacatggc agatttctcc 20 66 21 DNAArtificial sequence Forward primer for PCR amplification of NGA172 SSLPmarker in Arabidopsis thaliana subspecies 66 agctgcttcc ttatagcgtc c 2167 19 DNA Artificial sequence Reverse primer for PCR amplification ofNGA172 SSLP marker in Arabidopsis thaliana subspecies 67 catccgaatgccattgttc 19 68 21 DNA Artificial sequence Forward primer for PCRamplification of NGA126 SSLP marker in Arabidopsis thaliana subspecies68 gaaaaaacgc tactttcgtg g 21 69 22 DNA Artificial sequence Reverseprimer for PCR amplification of NGA126 SSLP marker in Arabidopsisthaliana subspecies 69 caagagcaat atcaagagca gc 22 70 20 DNA Artificialsequence Forward primer for PCR amplification of NGA162 SSLP marker inArabidopsis thaliana subspecies 70 catgcaattt gcatctgagg 20 71 22 DNAArtificial sequence Reverse primer for PCR amplification of NGA162 SSLPmarker in Arabidopsis thaliana subspecies 71 ctctgtcact cttttcctct gg 2272 21 DNA Artificial sequence Forward primer for PCR amplification ofNGA6 SSLP marker in Arabidopsis thaliana subspecies 72 tggatttcttcctctcttca c 21 73 21 DNA Artificial sequence Reverse primer for PCRamplification of NGA6 SSLP marker in Arabidopsis thaliana subspecies 73atggagaagc ttacactgat c 21 74 20 DNA Artificial sequence Forward primerfor PCR amplification of NGA12 SSLP marker in Arabidopsis thalianasubspecies 74 aatgttgtcc tcccctcctc 20 75 22 DNA Artificial sequenceReverse primer for PCR amplification of NGA12 SSLP marker in Arabidopsisthaliana subspecies 75 tgatgctctc tgaaacaaga gc 22 76 21 DNA Artificialsequence Forward primer for PCR amplification of NGA8 SSLP marker inArabidopsis thaliana subspecies 76 gagggcaaat ctttatttcg g 21 77 22 DNAArtificial sequence Reverse primer for PCR amplification of NGA8 SSLPmarker in Arabidopsis thaliana subspecies 77 tggctttcgt ttataaacat cc 2278 21 DNA Artificial sequence Forward primer for PCR amplification ofNGA1107 SSLP marker in Arabidopsis thaliana subspecies 78 gcgaaaaaacaaaaaaatcc a 21 79 21 DNA Artificial sequence Reverse primer for PCRamplification of NGA1107 SSLP marker in Arabidopsis thaliana subspecies79 cgacgaatcg acagaattag g 21 80 21 DNA Artificial sequence Forwardprimer for PCR amplification of NGA225 SSLP marker in Arabidopsisthaliana subspecies 80 gaaatccaaa tcccagagag g 21 81 22 DNA Artificialsequence Reverse primer for PCR amplification of NGA225 SSLP marker inArabidopsis thaliana subspecies 81 tctccccact agttttgtgt cc 22 82 19 DNAArtificial sequence Forward primer for PCR amplification of NGA249 SSLPmarker in Arabidopsis thaliana subspecies 82 taccgtcaat ttcatcgcc 19 8322 DNA Artificial sequence Reverse primer for PCR amplification ofNGA249 SSLP marker in Arabidopsis thaliana subspecies 83 ggatccctaactgtaaaatc cc 22 84 22 DNA Artificial sequence Forward primer for PCRamplification of CA72 SSLP marker in Arabidopsis thaliana subspecies 84aatcccagta accaaacaca ca 22 85 20 DNA Artificial sequence Reverse primerfor PCR amplification of CA72 SSLP marker in Arabidopsis thalianasubspecies 85 cccagtctaa ccacgaccac 20 86 20 DNA Artificial sequenceForward primer for PCR amplification of NGA151 SSLP marker inArabidopsis thaliana subspecies 86 gttttgggaa gttttgctgg 20 87 24 DNAArtificial sequence Reverse primer for PCR amplification of NGA151 SSLPmarker in Arabidopsis thaliana subspecies 87 cagtctaaaa gcgagagtat gatg24 88 22 DNA Artificial sequence Forward primer for PCR amplification ofNGA106 SSLP marker in Arabidopsis thaliana subspecies 88 gttatggagtttctagggca cg 22 89 20 DNA Artificial sequence Reverse primer for PCRamplification of NGA106 SSLP marker in Arabidopsis thaliana subspecies89 tgccccattt tgttcttctc 20 90 20 DNA Artificial sequence Forward primerfor PCR amplification of NGA139 SSLP marker in Arabidopsis thalianasubspecies 90 agagctacca gatccgatgg 20 91 21 DNA Artificial sequenceReverse primer for PCR amplification of NGA139 SSLP marker inArabidopsis thaliana subspecies 91 ggtttcgttt cactatccag g 21 92 22 DNAArtificial sequence Forward primer for PCR amplification of NGA76 SSLPmarker in Arabidopsis thaliana subspecies 92 ggagaaaatg tcactctcca cc 2293 20 DNA Artificial sequence Reverse primer for PCR amplification ofNGA76 SSLP marker in Arabidopsis thaliana subspecies 93 aggcatgggagacatttacg 20 94 20 DNA Artificial sequence Forward primer for PCRamplification of ATHSO191 SSLP marker in Arabidopsis thaliana subspecies94 ctccaccaat catgcaaatg 20 95 21 DNA Artificial sequence Reverse primerfor PCR amplification of ATHSO191 SSLP marker in Arabidopsis thalianasubspecies 95 tgatgttgat ggagatggtc a 21 96 22 DNA Artificial sequenceForward primer for PCR amplification of NGA129 SSLP marker inArabidopsis thaliana subspecies 96 tcaggaggaa ctaaagtgag gg 22 97 22 DNAArtificial sequence Reverse primer for PCR amplification of NGA129 SSLPmarker in Arabidopsis thaliana subspecies 97 cacactgaag atggtcttga gg 2298 8062 DNA Arabidopsis thaliana ecotype Columbia Genomic DNA sequenceof AtMSH6 98 ttttttggtt gctaacaata aaggtatacg gttttatgtc atcaatataactatatataa 60 aagaaatgaa agatatatat tgttttttca tttatcaaac aaaacaacaagacttttttt 120 ttacttttta cattggtcaa caaaatacaa gataaacgac atcgtttaatcatttcccaa 180 ttttacccct aagtttaaca cctagaacct tctccatctt cgcaagcacagcctgattag 240 gaacagcttt accattctca tattcctgaa ctacctgagt cctctcattgatctgtttcg 300 ccaaatccgc ttgtgacatc ttcttctcca atctcgcttt ctgtatcatcaacctcacct 360 ctgctttcac acgatccatc gccgcaggct ctgtttcttc ttccagcttcttcgtgttaa 420 tcaccggaac cgccgtagat ttcccctttt tgttcgaacc ggcatcgaatttcttaaccg 480 tttgaaccgc gacaccgttt ctcagagctg cgttaaccgc tttcggatcgcgtaggtctt 540 ggctcttttg ttttgatttg tggagaacta ctggttccca gtcttgtgttactgctcctg 600 ggtatctgct cggcatcgtc gatgaattga gagaaaggaa caacgcgaaaattttattaa 660 tctgagtttt gaaattgaga aacgatgaag atgaagaatg ttgttgagaggattgtgata 720 tttatatata cgaagattgg tttctggaga attcgatcat ctttttctccattttcgtct 780 ctggaacgtt cttagagatg attgacgacg tgtcattatc tgatttgcagttaaccaatg 840 ctttttgggt tggattcgtg gtacaccata ttatccgatt tggctcaatggttttatata 900 aatttggttt tcggttcggt tatgagttat cattaaaatt aagctaaccaaaaattttcg 960 taaaatttat ttcggtttca attcggatcc cttacttcca gaaccgaattattcgaaacc 1020 ggggttagcc gaaccgaata ccaatgcctg attgactcgt tggctagaaagatccaacgg 1080 tatacaataa tagaacataa atcggacggt catcaaagcc tcaaagagtgaacagtcaac 1140 aaaaaaagtt gagccctgag gagtatcgtt tccgccattt ctacgacgcaaggcgaaaat 1200 ttttggcgcc aatctttccc ccctttcgaa ttctctcagc tcaaaacatcgtttctctct 1260 cactctctct cacaattcca aaaaatgcag cgccagagat cgattttgtctttcttccaa 1320 aaacccacgg cggcgactac gaagggtttg gtttccggcg atgctgctagcggcgggggc 1380 ggcagcggag accacgattt aatgtgaagg aaggggatgc taaaggcgacgcttctgtac 1440 gttttgctgt ttcgaaatct gtcgatgagg ttagaggaac ggatactccaccggagaagg 1500 ttccgcgtcg tgtcctgccg tctggattta agccggctga atccgccggtgatgcttcgt 1560 ccctgttctc caatattatg cataagtttg taaaagtcga tgatcgagattgttctggag 1620 agaggtacta atcttcgatt ctcttaattt tgttatcttt agctggaagaagaagattcg 1680 tgtaatttgt tgtattcgtt ggagagattc tgattactgc attggatcgttgtttacaaa 1740 ttttcaggag ccgagaagat gttgttccgc tgaatgattc atctctatgtatgaaggcta 1800 atgatgttat tcctcaattt cgttccaata atggtaaaac tcaagaaagaaaccatgctt 1860 ttagtttcag tgggagagct gaacttagat cagtagaaga tataggagtagatggcgatg 1920 ttcctggtcc agaaacacca gggatgcgtc cacgtgcttc tcgcttgaagcgagttctgg 1980 aggatgaaat gacttttaag gaggataagg ttcctgtatt ggactctaacaaaaggctga 2040 aaatgctcca ggatccggtt tgtggagaga agaaagaagt aaacgaaggaaccaaatttg 2100 aatggcttga gtcttctcga atcagggatg ccaatagaag acgtcctgatgatccccttt 2160 acgatagaaa gaccttacac ataccacctg atgttttcaa gaaaatgtctgcatcacaaa 2220 agcaatattg gagtgttaag agtgaatata tggacattgt gcttttctttaaagtggtta 2280 gtaactatta atctagtgtt caatccattt cctcaatgtg atttgttcacttacatctgt 2340 ttacgttatg ctcttctcag gggaaatttt atgagctgta tgagctagatgcggaattag 2400 gtcacaagga gcttgactgg aagatgacca tgagtggtgt gggaaaatgcagacaggtaa 2460 attagttgaa acaactggcc tgcttgaatt attgtgtcta taaattttgacaccaccttt 2520 tgtttcaggt tggtatctct gaaagtggga tagatgaggc agtgcaaaagctattagctc 2580 gtgggtaagg gaaccatcat actttatgga attcgtttac tgctacttcggctaggattt 2640 aagaaatgga aatcacttca agcatcatta gttaggatcc tgagaactcaggatgttttc 2700 ttattcgtta tataataagt cttttcatca aggagtaaca aacaaaacttgcacaatatt 2760 tgtgtgctca ctggcaaggc atatataccc agctaacctt tgctagttcactgtagtaac 2820 agttacggat aatatatgtt tacttgtatg tggtaccctc attttgtctctcatggaggc 2880 tttcaagcct tgtgttgaaa ctggatagtt acatatgctt ccaacagaaactagcatgca 2940 gattcatatg ctttcctatt ctactaatta tgtattgaca cactcgttgtttcttttgaa 3000 agatataaag ttggacgaat cgagcagcta gaaacatctg accaagcaaaagccagaggt 3060 gctaatactg taagttttct tggataggtc aaggagagtg ttgcagactgtttttgatca 3120 tttctttttc tgtacattac tttcatgctg taattaactc aatggctattctggtctgat 3180 tatcagataa ttccaaggaa gctagttcag gtattaactc catcaacagcaagcgaggga 3240 aacatcgggc ctgatgccgt ccatcttctt gctataaaag aggtttgttatttacttatt 3300 tatcttatca tgttcagttc atccaagtcc tgaaaaatta cactcttctttaccaatctt 3360 ccatcaagct gtgtaaagga tttggaatta gaaaatcatt atttgatgctttgttttata 3420 tgcaagaggt tcccttgaaa agatctgttt aagattcttt gcacttgaaaaattcaatct 3480 ttttaagtga atcccctact ttcttacaat gatcatagtc tgcaattgcatgtcaagtaa 3540 tatcattcct tgttactgca tccccctctt tcttaatgac cattgtctatgttgtgtttg 3600 tctcgtgtgc tggagaaaat gatagctgat ccaagctgta cattatcatgattaagtagc 3660 tgctcaggaa ttgcctttgg ttacattgcc taatggtttg atgtcaatttttcttctgaa 3720 tctttatttt agatcaaaat ggagctacaa aagtgttcaa ctgtgtatggatttgctttt 3780 gttgactgtg ctgccttgag gttttgggtt gggtccatca gcgatgatgcatcatgtgct 3840 gctcttggag cgttattgat gcaggtaagc aagtgtattc tgtatcttatgtgtaccatg 3900 tgacttcctg tgcatatatt tgggttgcag gaactaattc tgaatcaccatttggtatgt 3960 tttttccagg tttctccaaa ggaagtgtta tatgacagta aaggtaaactgcttgtatcg 4020 ccagttgttt tgttaaacag aatttaaggt aaatgacact ggttaatttaaagtgcatac 4080 atgttgaaat attgcagggc tatcaagaga agcacaaaag gctctaaggaaatatacgtt 4140 gacaggtacc atttcagtag gcaagctaac tgacaattta accgctcaccgaatgatagg 4200 tctcttaaac attgctaatg tagatgatgt ttatgtttca atctaatagggtctacggcg 4260 gtacagttgg ctccagtacc acaagtaatg ggggatacag atgctgctggagttagaaat 4320 ataatagaat ctaacggata ctttaaaggt tcttctgaat catggaactgtgctgttgat 4380 ggtctaaatg aatgtgatgt tgcccttagt gctcttggag agctaattaatcatctgtct 4440 aggctaaagg tgtgttggct tgtttagttt ttgcttttca caaattaagcaaaggaactt 4500 ttcataactt acagtttcta tctacttgca gctagaagat gtacttaagcatggggatat 4560 ttttccatac caagtttaca ggggttgtct cagaattgat ggccagacgatggtaaatct 4620 tgagatattt aacaatagct gtgatggtgg tccttcaggc aagtgcatatttcttttttg 4680 ataacttcaa ctagagggca gacatagaag gaaaaattct aatacttcgtacggatctcc 4740 agtaagtaat agccgatttt tgtttaccta tgtagggacc ttgtacaaatatcttgataa 4800 ctgtgttagt ccaactggta agcgactctt aaggaattgg atctgccatccactcaaaga 4860 tgtagaaagc atcaataaac ggcttgatgt agttgaagaa ttcacggcaaactcagaaag 4920 tatgcaaatc actggccagt atctccacaa acttccagac ttagaaagactgctcggacg 4980 catcaagtct agcgttcgat catcagcctc tgtgttgcct gctcttctggggaaaaaagt 5040 gctgaaacaa cgagtaagta tcaatcacaa gttttctgag taatgccttccatgagtagt 5100 ataggactaa aacattacgg gtctagctaa agactgttct ccttcttttgcaatgtctgg 5160 ttattcatta catttctctt aacttattgc attgcaggtt aaagcatttgggcaaattgt 5220 gaaagggttc agaagtggaa ttgatctgtt gttggctcta cagaaggaatcaaatatgat 5280 gagtttgctt tataaactct gtaaacttcc tatattagta ggaaaaagcgggctagagtt 5340 atttctttct caattcgaag cagccataga tagcgacttt ccaaattatcaggtgcccat 5400 ctatctttca tactttacaa caaaatgtct gtcactactc aaagcaatgcatatggctta 5460 gatctcaact cacaccccga ggatcctaaa gggatttgct ttttattcctaatgtttttg 5520 gatggtttga tttatttcta acttgaactt attaatcttg taccagaaccaagatgtgac 5580 agatgaaaac gctgaaactc tcacaatact tatcgaactt tttatcgaaagagcaactca 5640 atggtctgag gtcattcaca ccataagctg cctagatgtc ctgagatcttttgcaatcgc 5700 agcaagtctc tctgctggaa gcatggccag gcctgttatt tttcccgaatcagaagctac 5760 agatcagaat cagaaaacaa aagggccaat acttaaaatc caaggactatggcatccatt 5820 tgcagttgca gccgatggtc aattgcctgt tccgaatgat atactccttggcgaggctag 5880 aagaagcagt ggcagcattc atcctcggtc attgttactg acgggaccaaacatgggcgg 5940 aaaatcaact cttcttcgtg caacatgtct ggccgttatc tttgcccaagtttgtatact 6000 cgttagataa ttactctatt ctttgcaatc agttcttcaa catgaataataaattctgtt 6060 ttctgtctgc agcttggctg ctacgtgccg tgtgagtctt gcgaaatctccctcgtggat 6120 actatcttca caaggcttgg cgcatctgat agaatcatga caggagagagtaagttttgt 6180 tctcaaaata ccaattcctc gaactattta ctcagatttt gtctgattggacaaggtggt 6240 tttgcttttt tttaggtacc tttttggtag aatgcactga gacagcgtcagttcttcaga 6300 atgcaactca ggattcacta gtaatccttg acgaactggg cagaggaactagtactttcg 6360 atggatacgc cattgcatac tcggtaacct gctcttctcc ttcaacttatacttgttgat 6420 caacaaaaac atgcaattca ttttgctgaa acttattgat ttatatcaggtttttcgtca 6480 cctggtagag aaagttcaat gtcggatgct ctttgcaaca cattaccaccctctcaccaa 6540 ggaattcgcg tctcacccac gtgtcacctc gaaacacatg gcttgcgcattcaaatcaag 6600 atctgattat caaccacgtg gttgtgatca agacctagtg ttcttgtaccgtttaaccga 6660 gggagcttgt cctgagagct acggacttca agtggcactc atggctggaataccaaacca 6720 agtggttgaa acagcatcag gtgctgctca agccatgaag agatcaattggggaaaactt 6780 caagtcaagt gagctaagat ctgagttctc aagtctgcat gaagactggctcaagtcatt 6840 ggtgggtatt tctcgagtcg cccacaacaa tgcccccatt ggcgaagatgactacgacac 6900 tttgttttgc ttatggcatg agatcaaatc ctcttactgt gttcccaaataaatggctat 6960 gacataacac tatctgaagc tcgttaagtc ttttgcttct ctgatgtttattcctcttaa 7020 aaaatgctta tatatcaaaa aattgtttcc tcgattataa caagattatatatgtatctg 7080 tcggtttagc tatggtatat aatatatgta tgttcatgag attggtcaagagaaatactc 7140 acaaacagta tattaagaag gaaatatgtt tatgcattaa tttaagtttcaagataaact 7200 gcaaataacc tcgactaaag ttgcaaagac caaacacaaa ttacaaaacttataagactt 7260 aagttctgaa ttccctaaaa ccaaaaaaaa aaacagaaca tattttgttgcatctacaaa 7320 caacacaaac ctacatagtt tataacttac tcatcactga gattaacatcagaatcattc 7380 tccatttctt catcttcact ctcatcatca tcaccaccac catgatgattctcctcctct 7440 tcacgtaacc tagcaatctc actctgagct ctatcaacaa tctgcttcttctgcaactcc 7500 aaatctctct gaaaatcagc tctcatcttc tccaactcct tcatttgctctttcttactc 7560 ttctccatct tctcataaac cttcccaaac ctctcaacag aatccgccaacatcttatac 7620 gaagcagcgt cattaacctt cttcctctcg tactcaacct catcatcctcatcctcctcc 7680 tcttcagaat caccaggact atccatcatc tcatcaaacc cattagacttatctaaataa 7740 accttagtgt tcataaacac aaactcacct gaatcaacac cacaagctaaacctaaatcc 7800 gacttgggcg aaacacaaag caacatatcc aacttattga aaaacgaccatttacttgaa 7860 cctaaacctg atttctcaac cttaatcttc tcttttctat acttcctcttcaagtcatca 7920 atcattctcc tacattgcgt ctcagatttc tccatcctta gctcctcactcactttctca 7980 gctacttcat tccaatcctc gttcctcaaa ctccttctac ccaattgcaaaaacctatct 8040 ccccaaactt caagcaacac aa 8062 99 5 PRT Arabidopsisthaliana PEPTIDE (1)...(5) Positions 816-820 of AtMSH3 and 852-856 ofAtMSH6 99 Thr Gly Pro Asn Met 1 5 100 5 PRT Arabidopsis thaliana PEPTIDE(1)...(5) Positions 964-968 of AtMSH6 100 Phe Ala Thr His Tyr 1 5 101 5PRT Arabidopsis thaliana PEPTIDE (1)...(5) Positions 928-932 of AtMSH3101 Phe Val Thr His Tyr 1 5 102 1047 PRT Saccharomyces cerevisiae 102Met Val Ile Gly Asn Glu Pro Lys Leu Val Leu Leu Arg Ala Lys Ser 1 5 1015 Ser Ala Asn Arg Phe Ile Leu Leu Asn Leu Leu Thr Ile Met Ala Gly 20 2530 Gln Pro Thr Ile Ser Arg Phe Phe Lys Lys Ala Val Lys Ser Glu Leu 35 4045 Thr His Lys Gln Glu Gln Glu Val Ala Val Gly Asn Gly Ala Gly Ser 50 5560 Glu Ser Ile Cys Leu Asp Thr Asp Glu Glu Asp Asn Leu Ser Ser Val 65 7075 80 Ala Ser Thr Thr Val Thr Asn Asp Ser Phe Pro Leu Lys Gly Ser Val 8590 95 Ser Ser Lys Asn Ser Lys Asn Ser Glu Lys Thr Ser Gly Thr Ser Thr100 105 110 Thr Phe Asn Asp Ile Asp Phe Ala Lys Lys Leu Asp Arg Ile MetLys 115 120 125 Arg Arg Ser Asp Glu Asn Val Glu Ala Glu Asp Asp Glu GluGlu Gly 130 135 140 Glu Glu Asp Phe Val Lys Lys Lys Ala Arg Lys Ser ProThr Ala Lys 145 150 155 160 Leu Thr Pro Leu Asp Lys Gln Val Lys Asp LeuLys Met His His Arg 165 170 175 Asp Lys Val Leu Val Ile Arg Val Gly TyrLys Tyr Lys Cys Phe Ala 180 185 190 Glu Asp Ala Val Thr Val Ser Arg IleLeu His Ile Lys Leu Val Pro 195 200 205 Gly Lys Leu Thr Ile Asp Glu SerAsn Pro Gln Asp Cys Asn His Arg 210 215 220 Gln Phe Ala Tyr Cys Ser PhePro Asp Val Arg Leu Asn Val His Leu 225 230 235 240 Glu Arg Leu Val HisHis Asn Leu Lys Val Ala Val Val Glu Gln Ala 245 250 255 Glu Thr Ser AlaIle Lys Lys His Asp Pro Gly Ala Ser Lys Ser Ser 260 265 270 Val Phe GluArg Lys Ile Ser Asn Val Phe Thr Lys Ala Thr Phe Gly 275 280 285 Val AsnSer Thr Phe Val Leu Arg Gly Lys Arg Ile Leu Gly Asp Thr 290 295 300 AsnSer Ile Trp Ala Leu Ser Arg Asp Val His Gln Gly Lys Val Ala 305 310 315320 Lys Tyr Ser Leu Ile Ser Val Asn Leu Asn Asn Gly Glu Val Val Tyr 325330 335 Asp Glu Phe Glu Glu Pro Asn Leu Ala Asp Glu Lys Leu Gln Ile Arg340 345 350 Ile Lys Tyr Leu Gln Pro Ile Glu Val Leu Val Asn Thr Asp AspLeu 355 360 365 Pro Leu His Val Ala Lys Phe Phe Lys Asp Ile Ser Cys ProLeu Ile 370 375 380 His Lys Gln Glu Tyr Asp Leu Glu Asp His Val Val GlnAla Ile Lys 385 390 395 400 Val Met Asn Glu Lys Ile Gln Leu Ser Pro SerLeu Ile Arg Leu Val 405 410 415 Ser Lys Leu Tyr Ser His Met Val Glu TyrAsn Asn Glu Gln Val Met 420 425 430 Leu Ile Pro Ser Ile Tyr Ser Pro PheAla Ser Lys Ile His Met Leu 435 440 445 Leu Asp Pro Asn Ser Leu Gln SerLeu Asp Ile Phe Thr His Asp Gly 450 455 460 Gly Lys Gly Ser Leu Phe TrpLeu Leu Asp His Thr Arg Thr Ser Phe 465 470 475 480 Gly Leu Arg Met LeuArg Glu Trp Ile Leu Lys Pro Leu Val Asp Val 485 490 495 His Gln Ile GluGlu Arg Leu Asp Ala Ile Glu Cys Ile Thr Ser Glu 500 505 510 Ile Asn AsnSer Ile Phe Phe Glu Ser Leu Asn Gln Met Leu Asn His 515 520 525 Thr ProAsp Leu Leu Arg Thr Leu Asn Arg Ile Met Tyr Gly Thr Thr 530 535 540 SerArg Lys Glu Val Tyr Phe Tyr Leu Lys Gln Ile Thr Ser Phe Val 545 550 555560 Asp His Phe Lys Met His Gln Ser Tyr Leu Ser Glu His Phe Lys Ser 565570 575 Ser Asp Gly Arg Ile Gly Lys Gln Ser Pro Leu Leu Phe Arg Leu Phe580 585 590 Ser Glu Leu Asn Glu Leu Leu Ser Thr Thr Gln Leu Pro His PheLeu 595 600 605 Thr Met Ile Asn Val Ser Ala Val Met Glu Lys Asn Ser AspLys Gln 610 615 620 Val Met Asp Phe Phe Asn Leu Asn Asn Tyr Asp Cys SerGlu Gly Ile 625 630 635 640 Ile Lys Ile Gln Arg Glu Ser Glu Ser Val ArgSer Gln Leu Lys Glu 645 650 655 Glu Leu Ala Glu Ile Arg Lys Tyr Leu LysArg Pro Tyr Leu Asn Phe 660 665 670 Arg Asp Glu Val Asp Tyr Leu Ile GluVal Lys Asn Ser Gln Ile Lys 675 680 685 Asp Leu Pro Asp Asp Trp Ile LysVal Asn Asn Thr Lys Met Val Ser 690 695 700 Arg Phe Thr Thr Pro Arg ThrGln Lys Leu Thr Gln Lys Leu Glu Tyr 705 710 715 720 Tyr Lys Asp Leu LeuIle Arg Glu Ser Glu Leu Gln Tyr Lys Glu Phe 725 730 735 Leu Asn Lys IleThr Ala Glu Tyr Thr Glu Leu Arg Lys Ile Thr Leu 740 745 750 Asn Leu AlaGln Tyr Asp Cys Ile Leu Ser Leu Ala Ala Thr Ser Cys 755 760 765 Asn ValAsn Tyr Val Arg Pro Thr Phe Val Asn Gly Gln Gln Ala Ile 770 775 780 IleAla Lys Asn Ala Arg Asn Pro Ile Ile Glu Ser Leu Asp Val His 785 790 795800 Tyr Val Pro Asn Asp Ile Met Met Ser Pro Glu Asn Gly Lys Ile Asn 805810 815 Ile Ile Thr Gly Pro Asn Met Gly Gly Lys Ser Ser Tyr Ile Arg Gln820 825 830 Val Ala Leu Leu Thr Ile Met Ala Gln Ile Gly Ser Phe Val ProAla 835 840 845 Glu Glu Ile Arg Leu Ser Ile Phe Glu Asn Val Leu Thr ArgIle Gly 850 855 860 Ala His Asp Asp Ile Ile Asn Gly Asp Ser Thr Phe LysVal Glu Met 865 870 875 880 Leu Asp Ile Leu His Ile Leu Lys Asn Cys AsnLys Arg Ser Leu Leu 885 890 895 Leu Leu Asp Glu Val Gly Arg Gly Thr GlyThr His Asp Gly Ile Ala 900 905 910 Ile Ser Tyr Ala Leu Ile Lys Tyr PheSer Glu Leu Ser Asp Cys Pro 915 920 925 Leu Ile Leu Phe Thr Thr His PhePro Met Leu Gly Glu Ile Lys Ser 930 935 940 Pro Leu Ile Arg Asn Tyr HisMet Asp Tyr Val Glu Glu Gln Lys Thr 945 950 955 960 Gly Glu Asp Trp MetSer Val Ile Phe Leu Tyr Lys Leu Lys Lys Gly 965 970 975 Leu Thr Tyr AsnSer Tyr Gly Met Asn Val Ala Lys Leu Ala Arg Leu 980 985 990 Asp Lys AspIle Ile Asn Arg Ala Phe Ser Ile Ser Glu Glu Leu Arg 995 1000 1005 LysGlu Ser Ile Asn Glu Asp Ala Leu Lys Leu Phe Ser Ser Leu Lys 1010 10151020 Arg Ile Leu Lys Ser Asp Asn Ile Thr Ala Thr Asp Lys Leu Ala Lys1025 1030 1035 1040 Leu Leu Ser Leu Asp Ile His 1045 103 1242 PRTSaccharomyces cerevisiae 103 Met Ala Pro Ala Thr Pro Lys Thr Ser Lys ThrAla His Phe Glu Asn 1 5 10 15 Gly Ser Thr Ser Ser Gln Lys Lys Met LysGln Ser Ser Leu Leu Ser 20 25 30 Phe Phe Ser Lys Gln Val Pro Ser Gly ThrPro Ser Lys Lys Val Gln 35 40 45 Lys Pro Thr Pro Ala Thr Leu Glu Asn ThrAla Thr Asp Lys Ile Thr 50 55 60 Lys Asn Pro Gln Gly Gly Lys Thr Gly LysLeu Phe Val Asp Val Asp 65 70 75 80 Glu Asp Asn Asp Leu Thr Ile Ala GluGlu Thr Val Ser Thr Val Arg 85 90 95 Ser Asp Ile Met His Ser Gln Glu ProGln Ser Asp Thr Met Leu Asn 100 105 110 Ser Asn Thr Thr Glu Pro Lys SerThr Thr Thr Asp Glu Asp Leu Ser 115 120 125 Ser Ser Gln Ser Arg Arg AsnHis Lys Arg Arg Val Asn Tyr Ala Glu 130 135 140 Ser Asp Asp Asp Asp SerAsp Thr Thr Phe Thr Ala Lys Arg Lys Lys 145 150 155 160 Gly Lys Val ValAsp Ser Glu Ser Asp Glu Asp Glu Tyr Leu Pro Asp 165 170 175 Lys Asn AspGly Asp Glu Asp Asp Asp Ile Ala Asp Asp Lys Glu Asp 180 185 190 Ile LysGly Glu Leu Ala Glu Asp Ser Gly Asp Asp Asp Asp Leu Ile 195 200 205 SerLeu Ala Glu Thr Thr Ser Lys Lys Lys Phe Ser Tyr Asn Thr Ser 210 215 220His Ser Ser Ser Pro Phe Thr Arg Asn Ile Ser Arg Asp Asn Ser Lys 225 230235 240 Lys Lys Ser Arg Pro Asn Gln Ala Pro Ser Arg Ser Tyr Asn Pro Ser245 250 255 His Ser Gln Pro Ser Ala Thr Ser Lys Ser Ser Lys Phe Asn LysGln 260 265 270 Asn Glu Glu Arg Tyr Gln Trp Leu Val Asp Glu Arg Asp AlaGln Arg 275 280 285 Arg Pro Lys Ser Asp Pro Glu Tyr Asp Pro Arg Thr LeuTyr Ile Pro 290 295 300 Ser Ser Ala Trp Asn Lys Phe Thr Pro Phe Glu LysGln Tyr Trp Glu 305 310 315 320 Ile Lys Ser Lys Met Trp Asp Cys Ile ValPhe Phe Lys Lys Gly Lys 325 330 335 Phe Phe Glu Leu Tyr Glu Lys Asp AlaLeu Leu Ala Asn Ala Leu Phe 340 345 350 Asp Leu Lys Ile Ala Gly Gly GlyArg Ala Asn Met Gln Leu Ala Gly 355 360 365 Ile Pro Glu Met Ser Phe GluTyr Trp Ala Ala Gln Phe Ile Gln Met 370 375 380 Gly Tyr Lys Val Ala LysVal Asp Gln Arg Glu Ser Met Leu Ala Lys 385 390 395 400 Glu Met Arg GluGly Ser Lys Gly Ile Val Lys Arg Glu Leu Gln Cys 405 410 415 Ile Leu ThrSer Gly Thr Leu Thr Asp Gly Asp Met Leu His Ser Asp 420 425 430 Leu AlaThr Phe Cys Leu Ala Ile Arg Glu Glu Pro Gly Asn Phe Tyr 435 440 445 AsnGlu Thr Gln Leu Asp Ser Ser Thr Ile Val Gln Lys Leu Asn Thr 450 455 460Lys Ile Phe Gly Ala Ala Phe Ile Asp Thr Ala Thr Gly Glu Leu Gln 465 470475 480 Met Leu Glu Phe Glu Asp Asp Ser Glu Cys Thr Lys Leu Asp Thr Leu485 490 495 Met Ser Gln Val Arg Pro Met Glu Val Val Met Glu Arg Asn AsnLeu 500 505 510 Ser Thr Leu Ala Asn Lys Ile Val Lys Phe Asn Ser Ala ProAsn Ala 515 520 525 Ile Phe Asn Glu Val Lys Ala Gly Glu Glu Phe Tyr AspCys Asp Lys 530 535 540 Thr Tyr Ala Glu Ile Ile Ser Ser Glu Tyr Phe SerThr Glu Glu Asp 545 550 555 560 Trp Pro Glu Val Leu Lys Ser Tyr Tyr AspThr Gly Lys Lys Val Gly 565 570 575 Phe Ser Ala Phe Gly Gly Leu Leu TyrTyr Leu Lys Trp Leu Lys Leu 580 585 590 Asp Lys Asn Leu Ile Ser Met LysAsn Ile Lys Glu Tyr Asp Phe Val 595 600 605 Lys Ser Gln His Ser Met ValLeu Asp Gly Ile Thr Leu Gln Asn Leu 610 615 620 Glu Ile Phe Ser Asn SerPhe Asp Gly Ser Asp Lys Gly Thr Leu Phe 625 630 635 640 Lys Leu Phe AsnArg Ala Ile Thr Pro Met Gly Lys Arg Met Met Lys 645 650 655 Lys Trp LeuMet His Pro Leu Leu Arg Lys Asn Asp Ile Glu Ser Arg 660 665 670 Leu AspSer Val Asp Ser Leu Leu Gln Asp Ile Thr Leu Arg Glu Gln 675 680 685 LeuGlu Ile Thr Phe Ser Lys Leu Pro Asp Leu Glu Arg Met Leu Ala 690 695 700Arg Ile His Ser Arg Thr Ile Lys Val Lys Asp Phe Glu Lys Val Ile 705 710715 720 Thr Ala Phe Glu Thr Ile Ile Glu Leu Gln Asp Ser Leu Lys Asn Asn725 730 735 Asp Leu Lys Gly Asp Val Ser Lys Tyr Ile Ser Ser Phe Pro GluGly 740 745 750 Leu Val Glu Ala Val Lys Ser Trp Thr Asn Ala Phe Glu ArgGln Lys 755 760 765 Ala Ile Asn Glu Asn Ile Ile Val Pro Gln Arg Gly PheAsp Ile Glu 770 775 780 Phe Asp Lys Ser Met Asp Arg Ile Gln Glu Leu GluAsp Glu Leu Met 785 790 795 800 Glu Ile Leu Met Thr Tyr Arg Lys Gln PheLys Cys Ser Asn Ile Gln 805 810 815 Tyr Lys Asp Ser Gly Lys Glu Ile TyrThr Ile Glu Ile Pro Ile Ser 820 825 830 Ala Thr Lys Asn Val Pro Ser AsnTrp Val Gln Met Ala Ala Asn Lys 835 840 845 Thr Tyr Lys Arg Tyr Tyr SerAsp Glu Val Arg Ala Leu Ala Arg Ser 850 855 860 Met Ala Glu Ala Lys GluIle His Lys Thr Leu Glu Glu Asp Leu Lys 865 870 875 880 Asn Arg Leu CysGln Lys Phe Asp Ala His Tyr Asn Thr Ile Trp Met 885 890 895 Pro Thr IleGln Ala Ile Ser Asn Ile Asp Cys Leu Leu Ala Ile Thr 900 905 910 Arg ThrSer Glu Tyr Leu Gly Ala Pro Ser Cys Arg Pro Thr Ile Val 915 920 925 AspGlu Val Asp Ser Lys Thr Asn Thr Gln Leu Asn Gly Phe Leu Lys 930 935 940Phe Lys Ser Leu Arg His Pro Cys Phe Asn Leu Gly Ala Thr Thr Ala 945 950955 960 Lys Asp Phe Ile Pro Asn Asp Ile Glu Leu Gly Lys Glu Gln Pro Arg965 970 975 Leu Gly Leu Leu Thr Gly Ala Asn Ala Ala Gly Lys Ser Thr IleLeu 980 985 990 Arg Met Ala Cys Ile Ala Val Ile Met Ala Gln Met Gly CysTyr Val 995 1000 1005 Pro Cys Glu Ser Ala Val Leu Thr Pro Ile Asp ArgIle Met Thr Arg 1010 1015 1020 Leu Gly Ala Asn Asp Asn Ile Met Gln GlyLys Ser Thr Phe Phe Val 1025 1030 1035 1040 Glu Leu Ala Glu Thr Lys LysIle Leu Asp Met Ala Thr Asn Arg Ser 1045 1050 1055 Leu Leu Val Val AspGlu Leu Gly Arg Gly Gly Ser Ser Ser Asp Gly 1060 1065 1070 Phe Ala IleAla Glu Ser Val Leu His His Val Ala Thr His Ile Gln 1075 1080 1085 SerLeu Gly Phe Phe Ala Thr His Tyr Gly Thr Leu Ala Ser Ser Phe 1090 10951100 Lys His His Pro Gln Val Arg Pro Leu Lys Met Ser Ile Leu Val Asp1105 1110 1115 1120 Glu Ala Thr Arg Asn Val Thr Phe Leu Tyr Lys Met LeuGlu Gly Gln 1125 1130 1135 Ser Glu Gly Ser Phe Gly Met His Val Ala SerMet Cys Gly Ile Ser 1140 1145 1150 Lys Glu Ile Ile Asp Asn Ala Gln IleAla Ala Asp Asn Leu Glu His 1155 1160 1165 Thr Ser Arg Leu Val Lys GluArg Asp Leu Ala Ala Asn Asn Leu Asn 1170 1175 1180 Gly Glu Val Val SerVal Pro Gly Gly Leu Gln Ser Asp Phe Val Arg 1185 1190 1195 1200 Ile AlaTyr Gly Asp Gly Leu Lys Asn Thr Lys Leu Gly Ser Gly Glu 1205 1210 1215Gly Val Leu Asn Tyr Asp Trp Asn Ile Lys Arg Asn Val Leu Lys Ser 12201225 1230 Leu Phe Ser Ile Ile Asp Asp Leu Gln Ser 1235 1240

1-36. (canceled)
 37. An isolated and purified nucleic acid moleculecomprising a nucleotide sequence encoding a polypeptide having the aminoacid sequence of AtMSH6 (SEQ ID NO:31).
 38. The nucleic acid of claim 37further comprising a regulation element operably linked to saidAtMSH6-encoding sequence.
 39. A plasmid or vector comprising the nucleicacid of claim
 38. 40. A plant cell stably transformed, transfected orelectroporated with the plasmid or vector of claim
 39. 41. A plantcomprising the cell of claim
 40. 42. A process for at least partiallyinactivating the DNA mismatch repair system of a plant cell comprising:transforming or transfecting said plant cell with a nucleic acidcomprising a regulation element operably linked to a nucleotide sequenceencoding a polypeptide having the amino acid sequence of AtMSH6 (SEQ IDNO:31); growing said cell under conditions that permit expression ofsaid AtMSH6-encoding sequence; and hereby inactivating the DNA mismatchrepair system of said plant cell.
 43. The process of claim 42, whereinsaid plant is selected from the group consisting of Brassicaceae,Poaceae, Solanaceae, Asteraceae, Malvaceae, Fabaceae, Linaceae,Canabinaceae, Dauaceae, and Curcubitaceae.
 44. A process for alteringthe mismatch repair system in a plant comprising introducing into saidplant a chimeric gene expressing a polynucleotide which is capable ofinterfering with the expression of the nucleotide sequence encoding aAtMSH6 polypeptide (SEQ ID NO:31).
 45. The process of claim 44, whereinsaid chimeric gene expresses a polynucleotide that is antisense withregard to the nucleic acid molecule comprising a nucleotide sequenceencoding said AtMSH6 polypeptide.